Chromatography matrices including novel Staphylococcus aureus protein A based ligands

ABSTRACT

The present invention relates to chromatography matrices including ligands based on one or more domains of immunoglobulin-binding proteins such as,  Staphylococcus aureus  Protein A (SpA), as well as methods of using the same.

RELATED APPLICATIONS

The present application is a Continuation Application of U.S.application Ser. No. 14/534,999, filed on Nov. 6, 2014 which is aContinuation Application of U.S. application Ser. No. 14/298,332, filedon Jun. 6, 2014, and which issued as U.S. Pat. No. 8,895,706, on Nov.25, 2014, which is a continuation of U.S. patent application Ser. No.13/489,999, filed on Jun. 6, 2012, and which issued as U.S. Pat. No.8,754,196 on Jun. 17, 2014, which claims the benefit of priority of U.S.Provisional Patent Application No. 61/494,701, filing date Jun. 8, 2011,each of which are incorporated by reference herein in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 31, 2015, isnamed MCA1353_(—)3US_SEQLIST.txt and is 124,391 bytes in size.

FIELD OF THE INVENTION

The present invention relates to chromatography matrices includingligands based on one or more domains of immunoglobulin-binding proteinssuch as Staphylococcus aureus Protein A (SpA) as well as methods ofusing the same.

BACKGROUND

Ligands used in affinity chromatography typically confer a highselectivity for the target molecule, thereby resulting in high yield,high parity and fast and economical purification of target molecules.Staphylococcus aureus Protein A-based reagents and chromatographymatrices have found a widespread use in the field of affinitychromatography for capture and purification of antibodies andFc-containing proteins as well as in analytical-scale antibody detectionmethods due to its ability to bind IgG, without significantly affectingthe affinity of the immunoglobulin for antigen.

Accordingly, various reagents and media comprising Protein A-ligandshave been developed and are commercially available, for example,ProSep®-vA High Capacity, ProSep® vA Ultra and ProSep® UltraPlus(MILLIPORE) and Protein A Sepharose™, MabSelect™, MabSelect Xtra™,MabSelect SuRe™ (GE HEALTHCARE), MabSelect SuRe™ LX and Poros MabCaptureA™ (LIFE TECHNOLOGIES).

In order to maintain selectivity of the chromatography ligands includingligand bound solid supports such as SpA bound chromatography matrices,matrices have to be cleaned and are typically cleaned under acidic oralkaline conditions, e.g., with sodium hydroxide (NaOH). For example, astandard process which is used for cleaning and restoring the matrix isa cleaning-in-place (CIP) alkaline protocol, which typically involvestreatment of the ligand bound matrix with NaOH concentration rangingfrom 0.05M to 1M, resulting in pH range 12.7 to 14.0. Typically,exposure of an affinity chromatography matrix to repeated CIP cyclesresults in significant loss of binding capacity of the matrix for atarget molecule over time, repairing the use of a greater amountthroughout the process, of often very expensive ligands which are boundto matrices. This is both uneconomical and undesirable as it results inthe purification process becoming more expensive as well as lengthy.

SUMMARY OF THE INVENTION

Protein A based chromatography matrices have been previously describedin the art which appear to show a reduced loss of binding capacity for atarget molecule following treatment with alkaline conditions. See, e.g.,U.S. Patent Publication No. 20100221844, which describes affinitychromatography matrices incorporating wild-type (wt) B or Z domains ofSpA with multiple point attachment to the matrix, which show up to 95%of the initial binding capacity even after exposure to 0.5M NaOH for 5hours or more. Also, U.S. Patent Publication No. 20100048876 describes achromatography matrix incorporating wild type C domain of SpA as well asa C domain containing a deletion of amino acid residues 3 through 6,which appear to show up to 95% of the initial binding capacity afterexposure to 0.5M for about 5 hours. These ligands are immobilized via acysteine directed single-point attachment to the matrix. Further,chromatography matrices have been described which incorporate Protein Adomains containing mutations at one or more asparagine residues of theprotein, where the matrices appear to show a reduced loss in bindingcapacity relative to the wild type SpA, following exposure to alkalineconditions and appear to be immobilized via a single-point attachment tothe matrix. See, e.g., U.S. Pat. No. 6,831,161.

Although, the aforementioned affinity chromatography matrices appear toshow a reduced loss in binding capacity for a target molecule followingexposure to caustic conditions, some of these matrices appear to show alarge degree of fragmentation of ligand, e.g., as observed usingSDS-PAGE and/or size exclusion chromatography (SEC), following exposureto caustic conditions. Such fragmentation is undesirable, as a largedegree of fragmentation of ligand results in smaller fragments ofligands being present which are more difficult to remove and separatefrom the target molecule, thereby increasing the likelihood that suchpotentially immunogenic fragments will co-purify with the therapeutictarget molecule. Furthermore, a large degree of fragmentation results inan increased loss in binding capacity of the matrix for a targetmolecule.

The present invention provides affinity chromatography ligands andmatrices incorporating the same, where the ligands are based on one ormore Staphylococcus aureus Protein A (SpA) domains having a deletionfrom the N-terminus, starting at position 1 or position 2 of the domain.These ligands and matrices show reduced fragmentation duringpurification use, as evidenced by SDS-PAGE and/or SEC techniques,relative to some of the previously described ligands, thereby makingthem more attractive and economical candidates for use in affinitychromatography.

In one aspect according to the present invention, affinitychromatography matrices are provided, which includes one or more Bdomains of SpA having a deletion, one or more C domains of SpA having adeletion of one or more Z domains of SpA having a deletion, where theone or more domains are attached to a solid support.

In one embodiment, an affinity chromatography matrix according to thepresent invention includes a ligand attached to a solid support, wherethe ligand comprises one or more B domains of Staphylococcus aureusProtein A (SpA), where at least one B domain comprises a deletion of atleast 3 consecutive amino acids from the N- In another embodiment, anaffinity chromatography matrix according to the present inventioncomprises a ligand attached to a solid support, where the ligandcomprises one or more C domains of Staphylococcus aureus Protein A(SpA), where at least one C domain comprises a deletion of at least 3consecutive amino acids from the N-terminus.

In yet another embodiment, an affinity chromatography matrix accordingto the present invention comprises a ligand attached to a solid support,where the ligand comprises one or more Z domains of Staphylococcusaureus Protein A (SpA), where at least one Z domain comprises a deletionof at least 3 consecutive amino acids from the N-terminus.

In still other embodiments, an affinity chromatography matrix accordingto the present invention comprises a ligand attached to a solid support,where the ligand comprises two or more B domains, two or more C domainsor two or more Z domains, or any combination of B, C and Z domains,where at least one of B, C or Z domain comprises a deletion of at least3 consecutive amino acids from the N-terminus.

In various embodiments according to the present invention, more than onesite on each ligand is attached to a solid support (i.e. multipointattachment).

In various embodiments according to the present invention, the ligandexhibits reduced fragmentation, as determined by SDS-PAGE or bysize-exclusion chromatography (SEC), relative to its wt counterpart,following exposure of the ligand or the matrix containing the ligand to0.5M NaOH for at least 5 hours.

In some embodiments according to the present invention, the ligandcomprises a deletion of 3 amino acids from the N-terminus, a deletion of4 amino acids from the N-terminus or a deletion of 5 amino acids fromthe N-terminus, where more than one site on the ligand is attached to asolid support, thereby to form an affinity chromatography matrix.

In a particular embodiment, a ligand has an amino acid sequence setforth in any of SEQ ID NOs:13-42, SEQ ID NOs:55-84 and SEQ ID NOs:93-94.

In another embodiment, a ligand according to the present invention hasthe following structure: [(X)_(n), (Y)_(m)]_(n+m), where X represents aB domain, a Z domain or a C domain of SpA, n represents the number ofdomains ranging from zero through (m−1), Y represents a B domain or a Zdomain or a C domain of SpA having at least 3 consecutive amino acidsdeleted from the N-terminus and m represents the number of Y domainsranging from one through eight, where more than one site on the ligandis attached to a solid support (e.g., a chromatography matrix).

In some embodiments according to the present invention, the ligandcomprises two B domains or two Z domains or two C domains of SpA, or oneB and one C domain, or one B and one Z domain, or one C and one Zdomain, where at least one B domain or at least one Z domain or at leastone C domain includes a deletion of three consecutive amino acids fromthe N-terminus or a deletion of four consecutive amino acids from theN-terminus or a deletion of five consecutive amino acids from theN-terminus. It is understood that the various domains may be arranged inany order.

In another embodiment, a ligand according to the present inventioncomprises three B domains or three Z domains or three C domains, or anycombination of B, C or Z domains in any order, where at least one Bdomain or at least one Z domain or at least one C domain comprises adeletion of three consecutive amino acids from the N-terminus or adeletion of tour consecutive amino acids from the N-terminus or adeletion of five consecutive amino acids from the N-terminus.

In yet another embodiment, a ligand according to the present inventioncomprises four B domains or four Z domains or four C domains, or anycombination of B, Z or C domains in any order, where at least one Bdomain or at least one Z domain or at least one C domain comprises adeletion of three consecutive amino acids from the N-terminus or adeletion of four consecutive amino acids from the N-terminus or adeletion of five consecutive amino acids from the N-terminus.

In yet another embodiment, a ligand according to the present inventioncomprises five B domains or five Z domains or five C domains, or anycombination of B, Z or C domains in any order, where at least one Bdomain or at least one Z domain or at least one C domain comprises adeletion of three consecutive amino-acids from the N-terminus or adeletion of four consecutive amino acids from the N-terminus or adeletion of five consecutive amino acids from the N-terminus.

In yet another embodiment, a ligand according to the present inventioncomprises six B domains or six Z domains or six C domains, or anycombination of B, Z or C domains in any order, where at least one Bdomain or at least one Z domain or at least one C domain comprises adeletion of three consecutive amino acids from the N-terminus or adeletion of four consecutive amino acids from the N-terminus or adeletion of five consecutive amino acids from the N-terminus.

In yet another embodiment, a ligand according to the present inventioncomprises seven B domains or seven Z domains or seven C domains, or anycombination of B, Z or C domains in any order, where at least one Bdomain or at least one Z domain or at least one C domain comprises adeletion of three consecutive amino acids from the N-terminus or adeletion of four consecutive amino acids from the N-terminus or adeletion of five consecutive amino acids from the N-terminus.

In a further embodiment, a ligand according to the present inventioncomprises eight B domains or eight Z domains or eight C domains, or anycombination of B, Z or C domains in any order, where at least one Bdomain or at least one Z domain or at least one C domain comprises adeletion of three consecutive amino acids from the N-terminus or adeletion of four consecutive amino acids from the N-terminus or adeletion of five consecutive amino acids from the N-terminus.

Additionally, provided herein are methods of using the affinitychromatography matrices. Accordingly, a method of affinity purifying oneor more target molecules (e.g., immunoglobulins or Fc-containingproteins) from a sample is provided, where the method comprises thesteps of: (a) providing a sample comprising one or more target molecules(e.g., immunoglobulins or Fc-containing proteins); (b) contacting thesample with a matrix according to the invention under conditions suchthat the one or more target molecules (e.g., immunoglobulins orFc-containing proteins) bind to the matrix; and (c) recovering the oneor more bound target molecules (e.g., immunoglobulins or Fc-containingproteins) by eluting under suitable conditions such as, for example, asuitable pH.

In some embodiments, an affinity chromatography matrix according to thepresent invention retains at least 95% of its initial binding capacityfor a target molecule after 5 hours, or after 10 hours, or after 15hours, or after 20 hours, or after 25 hours, or after 30 hours ofincubation in 0.5 M NaOH.

In a particular embodiment, an affinity chromatography matrix accordingto the present invention retains at least 95% of its initial bindingcapacity after 5 hours incubation in 0.5M NaOH.

In yet another embodiment an affinity chromatography matrix according tothe present invention retains at least 95% of its initial bindingcapacity for a target molecule alter 25 hours incubation in 0.1M NaOH;at least 85% of its initial binding capacity for a target molecule after25 hours incubation in 0.3M NaOH; or at least 65% of its initial bindingcapacity for a target molecule after 25 hours incubation in 0.5M NaOH.

The immunoglobulins which are capable of being bound by the variousligands described herein include, e.g., IgG, IgA and IgM, or any fusionprotein comprising an antibody and any fragment of antibody, which iscapable of binding to SpA.

Also provided herein am nucleic acid molecules encoding the variousligands described herein, as well as host cells including such nucleicacid molecules. In some embodiments, a host cell is a prokaryotic cell.In other embodiments, a host cell is a eukaryotic cell.

In some embodiments, the present, invention provides SpA-based affinitychromatography matrices which exhibit altered (increased or decreased)binding to a Fab portion of an immunoglobulin compared to the wt SpAligands, while retaining the ability to bind the Fc portion of theimmunoglobulin. In one embodiment, an SpA-based matrix according to thepresent invention exhibits a decreased binding to a Fab portion of animmunoglobulin compared to wt SpA. In a particular embodiment, achromatography matrix incorporates a SpA ligand, which includes a lysineat position 29, instead of a glycine (in case of B and C domains of SpA)or instead of an alanine (in case of the Z domain of SpA).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino-acid sequence alignments for the wild type (wt)IgG binding domains of SpA as well as the Z domain, represented by SEQID NOs:1-6.

FIG. 2 depicts schematic diagrams of the plasmid pET11a containing thenucleic acid sequence encoding the dimeric Z domain ligand with a A29Kmutation, the amino acid sequence shown in SEQ ID NO:85 (control), andplasmid pET11a containing the nucleic acid sequence encoding the dimericZ domain ligand with a A29K mutation as well as the second domainincluding a deletion of 4 consecutive amino acids from the N-terminus,the amino acid sequence shown in SEQ ID NO:78. The ligand constructsfurther include a His-tag sequence at the 3′ end.

FIG. 3 is a Coomassie stained SDS-PAGE gel for analyzing thefragmentation pattern of free and immobilized dimeric Z and C ligandswith or without caustic soak in 0.5M NaOH for 25 hrs. The description ofthe various Lanes of the SDS-PAGE gel is as follows. Lane 1: molecularmarker; Lane 2: dimeric Z domain ligand with no caustic exposure (A29Kwith no deletions, shown in SEQ ID NO:85, which is used as the controland includes a His-tag); Lane 3: the dimeric Z domain ligand controlsubjected to 0.5M NaOH soak for 25 hours; Lane 4: the dimeric Z domainligand control immobilized onto an agarose chromatography resin, whichis subjected to 0.5M NaOH soak for 25 hours; Lane 5: the dimeric Zdomain ligand having a deletion of 4 consecutive amino acids from theN-terminus of the second domain (A29K with the second domain having adeletion, shown in SEQ ID NO:78 and a His-tag) with no caustic exposure;Lane 6: the dimeric Z domain ligand of SEQ ID NO:78 with a His-tagsubjected to 0.5M NaOH soak for 25 hours; Lane 7: the dimeric Z domainligand of SEQ ID NO:78 with a His-tag immobilized onto an agarosechromatography resin and subjected to 0.5M NaOH soak for 25 hours; Lane8: dimeric C domain ligand with no deletions used as a control (aminoacid sequence shown in SEQ ID NO:92 plus having a His-tag) with nocaustic exposure; Lane 9: the dimeric C domain ligand control subjectedto 0.5M NaOH soak for 25 hours; Lane 10: the dimeric C domain ligandimmobilized onto an agarose chromatography resin and subjected to 0.5MNaOH soak for 25 hours; Lane 11: dimeric C domain ligand having adeletion from the N-terminus of the second domain (amino acid sequenceshown in SEQ ID NO:35 plus having a His-tag); Lane 12: dimeric C domainligand of SEQ ID NO:35 plus a His-tag subjected to 0.5M NaOH soak for 25hours; and Lane 13: dimeric C ligand of SEQ ID NO:35 plus a His-tagimmobilized onto an agarose chromatography resin and subjected to 0.5MNaOH soak for 25 hours.

FIG. 4 is a chromatogram of an SEC analysis of the dimeric Z and Cligands summarised in the description of FIG. 3 above. The x-axisdenotes the retention time in minutes with smaller molecules havinglonger retention time than that of a larger molecule. The y-axisrepresents UV absorption at 280 nm in mAU. The evidence of reducedfragmentation for the dimeric Z and C domain ligands having a N-terminusdeletion in the second domain, following extended caustic soak (i.e.,0.5M NaOH soak for 25 hours), is shown by way of boxes on thechromatogram and the presence of smaller fragments for the dimeric Z andC domain controls is shown by way of arrows.

FIG. 5 is a Coomassie stained SDS-PAGE gel for analyzing thefragmentation pattern of both free and immobilized pentameric Z domainligands with or without caustic soak in 0.5M NaOH for 25 hours. Thedescription of the various lanes of the SDS-PAGE gel is as follows: Lane1: molecular weight marker; Lane 2: pentameric Z domain ligand havingthe A29K mutation and a deletion of 4 consecutive amino acids from theN-terminus of all but the first domain, the amino acid sequence of whichis set forth in SEQ ID NO:84, with no caustic exposure; Lane 3:pentameric Z domain ligand of SEQ ID NO:84 subjected to 0.5M NaOH soakfor 25 hours; Lane 4: pentameric Z domain ligand of SEQ ID NO:84immobilized onto an agarose chromatography resin and subjected to 0.5MNaOH soak for 25 hours; Lane 5; pentameric Z domain ligand of SEQ IDNO:91, used as a control, which is not subjected to caustic soak; Lane6: pentameric Z domain ligand control subjected to 0.5M NaOH soak for 25hours; and Lane 7: pentameric Z domain ligand control immobilized ontoan agarose chromatography resin and subjected to 0.5M NaOH soak for 25hours. Further, Lanes 8, 9 and 10 relate to the results seen withsubjecting rSPA to a similar treatment, where Lane 8 represents rSPAwhich is not subjected to any caustic soak; Lane 9 represents rSPAsubjected to 0.5M NaOH soak for 25 hours and immobilized rSPA subjectedto 0.5M NaOH soak for 25 hours. The bands represent the fragmentation,as depicted by arrows.

FIG. 6 is a chromatogram of an SEC analysis of the pentameric Z domainligands summarized in the description of FIG. 3 above. The x-axisdenotes the retention time in minutes with smaller molecules havinglonger retention time than that of a larger molecule. The y-axisrepresents UV absorption at 280 nm in mAU. The evidence for reducedfragmentation in case of the pentameric Z domain ligands having aN-terminal deletion in all but the first domain, following extendedcaustic soak, is shown by way of boxes on the chromatogram and thepresence of smaller fragments seen with the pentameric Z domain controlis shown by way of an arrow pointing to the fragments. Further,extensive amount of fragmentation seen for rSPA can be also observedusing SEC.

FIG. 7 is a chromatogram of an SEC analysis of the immobilizedpentameric Z domain ligands summarized in the description of FIG. 5above. The x-axis denotes the retention time in minutes with smallermolecules having longer retention time than that of a larger molecule.The y-axis represents UV absorption at 280 nm in mAU. The evidence forreduced fragmentation in case of the immobilized pentameric Z domainligand having an N-terminal deletion in the second domain, followingextended caustic soak, is shown by way of a box on the chromatogram andthe presence of smaller fragments seen with the pentameric Z domaincontrol is shown by way of an arrow pointing to the fragments. Further,extensive amount of fragmentation seen for immobilized rSPA can be alsoobserved using SEC.

FIG. 8 is a chromatogram of an SEC analysis of the free dimeric Z domainligands following extended caustic soak, where the ligands include aN-terminus deletion of the first one (SEQ ID NO:87), first two SEQ IDNO:88), first three (SEQ ID NO:69) or first four (SEQ ID NO:78) of thesecond domain of the dimeric ligands. The x-axis denotes the retentiontime in minutes with smaller molecules having longer retention time thanthat of a larger molecule. The y-axis represents UV absorption at 280 nmin mAU. The evidence for reduced fragmentation in case of the dimericligands having first three or first four amino acids deleted from theN-terminus of the second domain following extended caustic soak isdepicted by boxes. The presence of fragmentation observed followingextended caustic soak of the dimeric ligands having no amino aciddeletions (SEQ ID NO:85) or the first amino acid deleted or the firsttwo amino acids deleted from the N-terminus of the second domain, isshown by way of arrows pointing to the presence of fragments on thechromatogram.

FIG. 9 compares the retained binding capacities of immobilized C domainpentameric ligands after repeated caustic exposure, where one pentamericligand includes an N terminus deletion of 4 amino acids in each domain,the G29K mutation in each domain as well as an alanine as the very firstamino acid in the pentamer (the amino acid sequence shown in SEQ IDNO:93); and the other pentameric ligand being its wt counterpart withthe G29 K mutation (the amino acid sequence of which is shown in SEQ IDNO:95). The x-axis represents time of cumulative exposure of thechromatography matrices to 0.7M NaOH over 16 cycles of 30 mins each. They-axis represents the percent retained binding capacity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides affinity chromatography matrices whichincorporate ligands based on one or more domains of SpA, where theligands, either alone or when immobilized onto a matrix, show reducedfragmentation during use in purification processes, relative to thecorresponding wt domains of SpA.

Previously described exemplary SpA-based chromatography ligands include,for example, those described in U.S. Patent Publication No. 20100221844,which describes chromatography matrices which incorporate wild type Band Z domains of SpA, where more than one site on the ligand is attachedto a chromatography matrix (i.e., multipoint attachment); thosedescribed in U.S. Patent Publication No. 20100048876, which discusseschromatography ligands based on the wt C domain of SpA, which arecapable of binding the Fab portions of some antibodies and are coupledto an insoluble carrier at a single site using a terminal couplinggroup; and those described in U.S. Pat. No. 6,831,161, which discussesSpA-based alkaline based chromatography ligands where one or moreasparagine amino acid residues have been modified.

As discussed above, while these ligands exhibit a reduced loss inbinding capacity following exposure to alkaline conditions, some ofthese ligands show fragmentation during use in purification process,e.g., the ligands described in U.S. Publication No. 20100221844, whichis highly undesirable. The ligands described herein, on the other hand,are far more attractive candidates for protein purification compared tothe previously described ligands, in that they show reducedfragmentation following exposure to alkaline conditions during theregeneration and cleaning-in-place (CIP) protocols that are routinelyused in protein purification processes.

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

1. Definitions

As used herein, the term “SpA,” “Protein A” or “Staphyloccus aureusProtein A,” refers to a 42 Kda multi-domain protein isolated from thebacterium Staphylococcus aureus. SpA is bound to the bacterial cell wallvia its carboxy-terminal cell wall binding region, referred to as the Xdomain. At the amino-terminal region, it includes fiveimmunoglobulin-binding domains, referred to as E, D, A, B, and C(Sjodhal, Eur J Biochem. September 78 (2):471-90 (1977); Uhlen et al., JBiol Chem. February 259 (3): 1695-702 (1984). Each of these domainscontains approximately 58 amino acid residues, and they share 65-90%amino acid sequence identity.

Each of the E, D, A, B and C domains of SpA possess distinct Ig-bindingsites. One site is for Fe□□ (the constant region of IgG class of Ig) andthe other is for the Fab portion of certain Ig molecules (the portion ofthe Ig that is responsible for antigen recognition). It has beenreported that each of the domains contains a Fab binding site. Thenon-Ig binding portion of SpA is located at the C-terminus and isdesignated the X region or X-domain.

The Z domain of SpA is an engineered analogue of the B domain of SpA andincludes a valine instead of an alanine at position 1 and an alanineinstead of a glycine residue at position 29 (Nilsson, et al., Proteinengineering, Vol 1, No. 2, 107-113, 1987.).

The cloning of the gene encoding SpA is described in U.S. Pat. No.5,151,350, the entire contents of which are incorporated by referenceherein in their entirety.

The present invention provides affinity chromatography matrices whichincorporate SpA-based ligands, where the ligands (both free as well asimmobilized ligands) exhibit reduced fragmentation, as observed bySDS-PAGE and SEC, following regeneration and CIP protocols that areroutinely used during protein purification process.

In some aspects according to the present invention, an affinity ligandcomprises one or more B domains or one or more Z domains or one or moreC domains, or any combinations thereof, where at least one B domain orat least one Z domain or at least one C domain comprises a deletion of 3consecutive amino acids from the N-terminus, or 4 consecutive aminoacids from the N-terminus or 5 consecutive amino acids from theN-terminus, starting at position 1 or at position 2.

In some embodiments according to the present invention, more than onesite of an affinity ligand is attached to a chromatography matrix (i.e.,multipoint attachment). In a particular embodiment, the presentinvention provides an affinity chromatography matrix comprising one ormore B domains of SpA attached to a chromatography matrix, where morethan one site of the ligand is attached to the matrix and where at leastone B domain has a deletion of 3 consecutive amino acids from theN-terminus or 4 consecutive amino acids from the N-terminus or 5consecutive amino acids from the N-terminus, starting at position 1 orat position 2 of the wt B domain sequence.

In another embodiment, the present invention provides an affinitychromatography matrix comprising one or more Z domains of SpA attachedto a chromatography matrix where more than one site of the ligand isattached to the matrix and where at least one Z domain has a deletion of3 consecutive amino acids from the N-terminus or 4 consecutive aminoacids from the N-terminus or 5 consecutive amino acids from theN-terminus, starting at position 1 or position 2 of the wt Z domainsequence.

In yet another embodiment, the present invention provides an affinitychromatography matrix comprising one or more C domains of SpA, attachedto a chromatography matrix, where more than one site of the ligand isattached to the matrix and where at least one C domain has a deletion of3 consecutive amino acids from the N-terminus or 4 consecutive aminoacids from the N-terminus or 5 consecutive amino acids from theN-terminus, starting at position 1 or at position 2 of the wt C domainsequence.

In a particular embodiment, the present invention provides an alkalinestable affinity chromatography ligand which includes five C domains ofSpA, with each domain including a G29K mutation as well as 4 amino acidsdeleted from the N-terminus, starting at position 1, and the pentamericform including an extra alanine as the first amino acid to facilitatehomogeneous post translational processing of the protein.

In some embodiments, SpA ligands described herein further include theglycine amino acid residue at position 29 replaced with a lysine aminoacid residue (in case of B and C domains) or the alanine amino acidresidue at position 29 replaced with a lysine amino acid residue (incase of Z domain).

The term “parental molecule” or “wild-type (wt) counterpart” or “wtprotein” or “wt domain,” as used herein, is intended to refer to acorresponding protein (SpA) or a domain of a protein (e.g., B, Z or Cdomains of SpA) in its substantially native form, which is generallyused as a control herein. A wt counterpart control, as used herein,which corresponds to a SpA domain in its substantially native form mayinclude one amino acid change from the corresponding SpA domain to alterFab binding: however, is otherwise identical in sequence to thecorresponding wt domain. The ligands according to the present inventionexhibit reduced fragmentation (in case of both free and immobilizedforms) relative to their wt counterparts (i.e., completely wt orincluding a mutation to alter Fab binding), as evidenced by theexperiments discussed in the Examples herein. In various embodiments,the wt counterpart of a B domain or C domain based ligand according tothe present invention is the wt B domain of SpA or wt C domain of SpA,the amino acid sequences of which are set forth in SEQ ID NO:3 and SEQID NO:4, respectively. In certain embodiments, a wt counterpart of a Zdomain based ligand is the Z domain amino acid sequence set forth in SEQID NO:6. In certain embodiments, a wt counterpart of a B, C or Z domain,is substantially identical to the sequence of the B, C or Z domainmentioned above, but for a mutation at position 29 to alter theFab-binding of the domain. Accordingly, in certain embodiments, a wtcounterpart of a B domain based ligand includes the amino acid sequenceset forth in SEQ ID NO:45 (G29K), a wt counterpart of a C domain basedligand includes the amino acid sequence set forth in SEQ ID NO:46 (G29K)and the wt counterpart of a Z domain based ligand includes the aminoacid sequence set forth in SEQ ID NO:48 (A29K). Further, in case aligand according to the present invention includes more than one domain,the corresponding wt counterpart will include the same number ofdomains; however, may include a mutation to alter Fab binding.Accordingly, in certain embodiments, a wt counterpart of a pentameric Cdomain ligand according to the present invention includes the amino acidsequence set forth in SEQ ID NO:95 or in SEQ ID NO:96.

The term “sequence identity” means that two nucleotide or amino acidsequences, when optimally aligned, such as by the programs GAP orBESTFIT using default gap weights, share at least 70% sequence identity,or at least 80% sequence identity, or at least 85% sequence identity, orat least 90% sequence identity, or at least 95% sequence identity ormore. For sequence comparison, typically one sequence acts as areference sequence (e.g., parent sequence), to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math, 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., Current Protocols in Molecular Biology). One example ofalgorithm that is suitable for determining percent sequence identity andsequence similarity is the BLAST algorithm, which is described inAltschul et al., J. Mol. Biol. 215:403 (1990). Software for performingBLAST analyses is publicly available through the National Center forBiotechnology information (publicly accessible through the NationalInstitutes of Health NCBI internet server). Typically, default programparameters can be used to perform the sequence comparison, althoughcustomized parameters can also be used. For amino acid sequences, theBLASTP program uses as defaults a wordlength (W) of 3, an expectation(E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff,Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

As used interchangeably herein, the terms “E domain,” “E domain of SpA,”and “E domain of Staphylococcus aureus Protein A,” refer to thepolypeptide whose amino acid sequence is set forth in SEQ ID NO:1 orthat encoded by, e.g., the nucleotide sequence set forth in SEQ ID NO:7.The “E domain” is a 51 amino acid polypeptide that folds into athree-helix bundle structure. It is capable of binding Fc via residueson the surface of helices 1 and 2, or to Fab via residues on the surfaceof helices 2 and 3.

As used interchangeably herein, the terms “D domain,” “D domain of SpA,”and “D domain of Staphylococcus aureus Protein A,” refer to thepolypeptide whose amino acid sequence is set forth in SEQ ID NO:5 orthat encoded by e.g., the nucleotide sequence set forth in SEQ ID NO:11.The “D domain” is a 61 amino acid polypeptide that folds into athree-helix bundle structure. It is capable of Fc binding via residueson the surface of helices 1 and 2, or to Fab via residues on the surfaceof helices 2 and 3.

As used interchangeably herein, the terms “A domain,” “A domain of SpA,”and “A domain of Staphylococcus aureus Protein A,” refer to thepolypeptide whose amino acid sequence is set forth in SEQ ID NO:2 orthat encoded by, e.g., the nucleotide sequence set forth in SEQ ID NO:8.The “A domain” is a 58 amino acid polypeptide that folds into athree-helix bundle structure. It is capable of Fc binding via residueson the surface of helices 1 and 2, or to Fab via residues on the surfaceof helices 2 and 3.

As used interchangeably herein, the terms “B domain,” “B domain of SpA,”and “B domain of Staphylococcus aureus Protein A,” refer to thepolypeptide whose amino acid sequence is set forth in SEQ ID NO:3 orthat encoded by, e.g., the nucleotide sequence set forth in SEQ ID NO:9.The “B domain” is a 58 amino acid polypeptide that folds into athree-helix bundle structure. It is capable of Fc binding via residueson the surface of helices 1 and 2, or to Fab via residues on the surfaceof helices 2 and 3.

In some embodiments, a B domain based ligand according to the presentinvention includes a deletion of three amino acids from the N-terminus,e.g., having the amino acid sequence set forth in SEQ ID NO:13. In otherembodiments, a B domain based ligand according to the present inventionincludes a deletion of four amino acids from the N-terminus, e.g.,having the amino acid sequence set forth in SEQ ID NO:28. In anotherembodiment, a B domain based ligand according to the present inventionincludes a deletion of five amino acids from the N-terminus (sequencenot shown).

As used interchangeably herein, the terms “C domain,” “C domain of SpA,”and “C domain of Staphylococcus aureus Protein A,” refer to thepolypeptide whose amino acid sequence is set forth in SEQ ID NO:4 orthat encoded by, e.g., the nucleotide sequence set forth in SEQ IDNO:10. The “C domain” is a 58 amino acid polypeptide that folds into athree-helix bundle structure. It is capable of Fc binding via residueson the surface of helices 1 and 2, or to Fab via residues on the surfaceof helices 2 and 3.

In some embodiments, a C domain based ligand according to the presentinvention includes a deletion of three amino acids from the N-terminus,e.g., having the amino acid sequence set forth in SEQ ID NO:14. In otherembodiments, a C domain based ligand according to the present inventionincludes a deletion of four amino acids from the N-terminus, e.g.,having the amino acid sequence set forth in SEQ ID NO:29. In anotherembodiment, a C domain based ligand according to the present inventionincludes a deletion of five amino acids from the N-terminus (sequencenot shown).

As used interchangeably herein, the terms “Z domain,” “Z domain of SpA”and “Z domain of Protein A,” refer to the three helix, 58 amino acidpolypeptide that is a variant of the B domain of protein A. The aminoacid sequence of the Z domain is set forth in SEQ ID NO:6 and thenucleic acid sequence is set forth in SEQ ID NO:12. An exemplary Zdomain is described in Nilsson et al., Protein Engr., 1:107-113 (1987),the entire contents of which are incorporated by reference herein.

In some embodiments, a Z domain based ligand according to the presentinvention includes a deletion of three amino acids from the N-terminus,e.g., having the amino acid sequence set forth in SEQ ID NO:15. In otherembodiments, a Z domain based ligand according to the present inventionincludes a deletion of four amino acids from the N-terminus, e.g.,having the amino acid sequence set forth in SEQ ID NO:30. In anotherembodiment, a Z domain based ligand according to the present inventionincludes a deletion of five amino acids from the N-terminus (sequencenot shown).

In some embodiments, more than one site of the ligands described hereinis attached to a solid support (i.e., multipoint attachment) and wherethe ligands show reduced fragmentation (in case of both free andattached ligands) during use in purification processes, as evidenced bySDS-PAGE or SEC.

The term “reduced fragmentation,” as used herein, refers to a decreasein the number and/or intensity of fragments of a ligand, as seen on anSDS-PAGE gel or by SEC, relative to a wt counterpart of the ligand,following exposure of the free ligand molecule or the ligand moleculeimmobilized onto a solid support, to alkaline conditions during thepurification process. In some embodiments, the ligand is immobilizedonto a solid support via multipoint attachment. Fragmentation is usuallydetected by the presence of lower molecular bands relative to the intactmolecule on an SDS-PAGE gel or as distinct peaks having differentretention times on an SEC chromatogram.

The SpA ligands according to the present invention exhibit reducedfragmentation, which can be detected as follows. For example, the freeligand can be exposed directly to 0.1M NaOH, 0.3M NaOH or 0.5M NaOH for25 hrs, followed by a pH adjustment to 7.0 and can be subsequentlyanalyzed by SDS-PAGE or SEC using standard protocols. Alternatively, aligand immobilized onto a chromatography matrix can be exposed to 0.1MNaOH, 0.3M NaOH or 0.5M NaOH for 25 hrs. The caustic supernatant issubsequently separated from the matrix (e.g., a resin) and neutralizedto pH 7. This supernatant can then be analyzed by SDS-PAGE or by SECusing standard protocols. In the case of SDS-PAGE, the relative opticalintensity of the fragments can be observed visually and compared to asuitable control (e.g., a wt domain of SpA or an SpA domain containing amutation at position 29, as described herein). In the case of SEC, therelative peak intensity can be observed and compared to a suitablecontrol (e.g., a wt domain of SpA or an SpA domain containing a mutationat position 29, as described herein).

A typical purification process using an affinity chromatography matrixinvolves regeneration of the matrix after each cycle of use employing anacidic or an alkaline solution, the latter being preferable. Inaddition, typical processes also involve CIP steps, which employ use ofan acidic or alkaline solution to sanitize the matrix, alkalinesolutions being preferable. Accordingly, an affinity chromatographymatrix is expected to be exposed to several cycles of regeneration andCIP steps in its lifetime, thereby resulting in a significant loss inbinding capacity for a target molecule over time.

The chromatography matrices incorporating the ligands according to thepresent invention are alkaline stable in addition to exhibiting reducedfragmentation during use in purification processes, in that they show areduced loss of binding capacity for a target molecule over time,following extended exposure to alkaline conditions during regenerationand CIP steps.

The term “alkaline-stable,” “alkaline stability,” “caustic stable” or“caustic stability,” as used herein, generally refers to the ability ofan affinity ligand according to the present invention, either alone orwhen immobilized onto a chromatography matrix, to withstand repeatedregeneration and CIP cycles using alkaline wash without losing itsinitial binding capacity. In general, it is assumed that a matrix byitself, onto which a ligand according to the invention is immobilized,contributes to less than a 5% change in stability after having beensoaked in 0.5M NaOH for up to 30 hours. For example, in someembodiments, affinity ligands according to the invention are able towithstand conventional alkaline cleaning for a prolonged period of time,which renders the ligands attractive candidates, especially forcost-effective large-scale purification of immunoglobulins andFc-containing proteins, many of which are therapeutic molecules.

In some embodiments, alkaline stability refers to the ability of aligand according to the present invention or a matrix incorporating aligand according to the present invention, to retain at least 65%, or atleast 70%, or at least 75%, or at least 80%, or at least 85%, or atleast 90%, or at least 95% of its initial binding capacity after 5hours, or after 10 hours, or after 15 hours, or after 20 hours, or after25 hours, or after 30 hours of incubation in 0.05M NaOH, 0.1M NaOH, 0.3MNaOH or 0.5M NaOH. In another embodiment, alkaline stability refers to adecrease in the initial binding capacity of the ligand by less than 70%,or less than 60%, or less than 50%, or less than 40%, or less than 30%even after treatment with 0.05M NaOH, 0.1M NaOH, 0.3M NaOH or 0.5M NaOHfor 5 hours or 7.5 hours or 10 hours or 15 hours or 20 hours or 25 hoursor 30 hours. In a particular embodiment, a chromatography matrixincorporating a ligand according to the present invention retains up to95% of its initial binding capacity after exposure to 0.5M NaOH for 5hours. In another embodiment, a chromatography matrix incorporating aligand according to the present invention retains up to 95% of itsinitial binding capacity after exposure to 0.1M NaOH for 25 hours. Inyet another embodiment, a chromatography matrix incorporating a ligandaccording to the present invention retains up to 85% of its initialbinding capacity after exposure to 0.3M NaOH for 25 hours. In a furtherembodiment, a chromatography matrix incorporating a ligand according tothe present invention retains up to 65% of its initial binding capacityafter exposure to 0.5M NaOH for 25 hours.

In some embodiments, SpA-based chromatography matrices according to thepresent invention exhibit an increased or improved alkaline stability ascompared to matrices including wild type SpA domains. However, in otherembodiments, SpA-based chromatography matrices according to the presentinvention are not more alkaline stable than the matrices includingwild-type counterparts of the ligands. One such example is a ligandbased on the pentameric C domain of SpA which is not more alkalinestable than its wild-type pentameric C domain counterpart. It isunderstood that in certain instances, both the wild-type and thevariants of SpA domains may include a G29K mutation to reduce Fabbinding; however, such mutation does not itself have an effect onalkaline stability (data not shown).

Alkaline stability can be readily measured by one of ordinary skill inthe art using routine experimentation and/or as described herein.

The term “initial binding capacity,” as used herein, refers to theamount of a target molecule (e.g., an immunoglobulin or an Fc-containingprotein) that can be captured by a unit volume of an affinitychromatography matrix (i.e., a matrix including an affinity ligand)prior to exposure of the matrix to alkaline conditions.

In some embodiments according to the present invention, affinitychromatography matrices including the ligands described herein (i.e.,containing one or more SpA B, Z or C domains including N-terminaldeletions described herein) exhibit less than 5%, or less than 6%, orless than 7%, or less than 8%, or less than 9%, or less than 10%, orless than 12%, or less than 15%, or less than 17%, or less than 20%, orless than 25%, or less than 30% loss in the initial binding capacity ofa target molecule relative to an affinity chromatography matrixcontaining a corresponding wt SpA domain counterpart, as describedherein, following extended exposure to caustic conditions. In someembodiments, the affinity chromatography matrices according to thepresent invention retain at least 95%, or at least 90%, or at least 85%,or at least 80%, or at least 75%, or at least 70% of the initial bindingcapacity of a target molecule relative to an affinity chromatographymatrix containing a corresponding wt SpA domain counterpart, followingextended exposure to caustic conditions. However, in some otherembodiments, the chromatography matrices according to the presentinvention exhibit similar binding capacity to matrices containing wtcounterpart of the ligand following extended exposure to causticconditions. One such exemplary chromatography matrix includes a ligandthat includes 5 or more C domains of SpA, where each domain includes 4amino acids deleted from the N-terminus where the ligand is not morealkaline stable than its wild-type C domain counterpart. Both thedeletion form and the wild-type counterpart may contain a G29K mutation.Further, in various embodiments described herein, the SpA ligands mayfurther include a single amino acid such as, an alanine, a valine or aglycine, at the N-terminus of only the first domain in a multimer, wherethe extra amino acid facilitates homogeneous post-translationalprocessing.

The binding capacity of an affinity chromatography ligand for a targetmolecule can be readily measured using methods known in the art andthose described herein., e.g., as described in U.S. Patent PublicationNo. 20100221844, incorporated by reference herein in its entirety.

The term “chromatography,” as used herein, refers to a dynamicseparation technique which separates a target molecule of interest(e.g., an immunoglobulin or an Fc-containing protein) from othermolecules in the mixture and allows it to be isolated. Typically, in achromatography method, a mobile phase (liquid or gas) transports asample containing the target molecule of interest across or through astationary phase (normally solid) medium. Differences in partition oraffinity to the stationary phase separate the different molecules whilemobile phase carries the different molecules out at different time.

The term “affinity chromatography,” as used herein, refers to a mode ofchromatography where a target molecule to be separated is isolated byits interaction with a molecule (e.g., an alkaline stable chromatographyligand) which specifically interacts with the target molecule. In oneembodiment, affinity chromatography involves the addition of a samplecontaining a target molecule (e.g., an immunoglobulin or anFc-containing protein) to a solid support which carries on it anSpA-based ligand, as described herein.

The term “Protein A affinity chromatography,” as used herein, refers tothe separation or isolation of substances using Protein A or SpA-basedligands, such as those described herein, where the SpA or Protein Aligand is immobilized, e.g., on a solid support. Examples of Protein Aaffinity chromatography media/resin known in the art include thosehaving the Protein A immobilised onto a controlled pore glass backbone,e.g., PROSEP A™ and PROSEP vA™ media/resin (MILLIPORE); those havingProtein A immobilized onto a polystyrene solid phase, e.g., the POROS50A™ and Poros MabCapture A™ media/resin (APPLIED BIOSYSTEMS, INC.); andthose having Protein A immobilized on an agarose solid support, e.g.,rPROTEIN A SEPHAROSE FAST FLOW™ or MABSELECT™ media or resins (GEHEALTHCARE).

In addition to the aforementioned matrices, Protein A may also beimmobilized onto a hydrophilic crosslinked polymer. See, e.g., U.S.Patent Publication No. 20080210615, incorporated by reference herein inits entirety, which describes exemplary hydrophilic crosslinkedpolymers. Without wishing to be bound by theory, it is contemplated thatthe ligands encompassed by the present invention may be immobilized ontohydrophilic crosslinked polymers, such as those described in U.S. PatentPublication No. 20080210615.

The term “affinity matrix” or “affinity chromatography matrix,” as usedinterchangeably herein, refers to a chromatographic support onto whichan affinity chromatography ligand (e.g., SpA or a domain thereof) isattached. The ligand is capable of binding to a molecule of interestthrough affinity interaction (e.g., an immunoglobulin or anFc-containing protein) which is to be purified or removed from amixture. Exemplary Protein A based affinity chromatography matrices foruse in Protein A based affinity chromatography which are known in theart include Protein A immobilized onto a controlled pore glass backbone,e.g., the PROSEP A™ and PROSEP vA™ resins, High Capacity, Ultra andPROSEP Ultra Plus (MILLIPORE); Protein A immobilized on a polystyrenesolid phase, e.g. the POROS 50A™ resin and POROS MabCapture A™ (APPLIEDBIOSYSTEMS); or Protein A immobilized on an agarose solid phase, forinstance the rPROTEIN A SEPHAROSE FAST FLOW™ or MABSELECT™ resin (GEHEALTHCARE).

The term “immunoglobulin,” “Ig” or “antibody” (used interchangeablyherein) refers to a protein having a basic four-polypeptide chainstructure consisting of two heavy and two light chains, said chainsbeing stabilized, for example, by interchain disulfide bonds, which hasthe ability to specifically bind antigen. The term “single-chainimmunoglobulin” or “single-chain antibody” (used interchangeably herein)refers to a protein having a two-polypeptide chain structure consistingof a heavy and a light chain, said chains being stabilized, for example,by interchain peptide linkers, which has the ability to specificallybind antigen. The term “domain” refers to a globular region of a heavyor light chain polypeptide comprising peptide loops (e.g., comprising 3to 4 peptide loops) stabilized, for example, by β-pleated sheet and/orintrachain disulfide bond. Domains are further referred to herein as“constant” or “variable”, based on the relative lack of sequencevariation within the domains of various class members in the case of a“constant” domain, or the significant variation within the domains ofvarious class members in the case of a “variable” domain. Antibody orpolypeptide “domains” are often referred to interchangeably in the artas antibody or polypeptide “regions”. The “constant” domains of antibodylight chains are referred to interchangeably as “light chain constantregions”, “light chain constant domains”, “CL” regions or “CL” domains.The “constant” domains of antibody heavy chains are referred tointerchangeably as “heavy chain constant regions”, “heavy chain constantdomains”, “CH” regions or “CH” domains. The “variable” domains ofantibody light chains are referred to interchangeably as “light chainvariable regions”, “light chain variable domains”, “VL” regions or “VL”domains. The “variable” domains of antibody heavy chains are referred tointerchangeably as “heavy chain variable regions”, “heavy chain variabledomains”, “VH” regions or “VH” domains.

Immunoglobulins or antibodies may be monoclonal or polyclonal and mayexist in monomeric or polymeric form, for example, IgM antibodies whichexist in pentameric form and/or IgA antibodies which exist in monomeric,dimeric or multimeric form. The term “fragment” refers to a part orportion of an antibody or antibody chain comprising fewer amino acidresidues than an intact or complete antibody or antibody chain.Fragments can be obtained via chemical or enzymatic treatment of anintact or complete antibody or antibody chain. Fragments can also beobtained by recombinant means. Exemplary fragments include Fab, Fab′,F(ab′)2, Fc and/or Fv fragments.

The term “antigen-binding fragment” refers to a polypeptide portion ofan immunoglobulin or antibody that binds an antigen or competes withintact antibody (i.e., with the intact antibody from which they werederived) for antigen binding (i.e., specific binding). Binding fragmentscan be produced by recombinant DNA techniques, or by enzymatic orchemical cleavage of intact immunoglobulins. Binding fragments includeFab, Fab′, F(ab′)₂, Fv, single chains, and single-chain antibodies.

Also encompassed are fusion proteins including an antibody or fragmentthereof as a part of the fusion protein.

The terms “polynucleotide” and “nucleic acid molecule,” usedinterchangeably herein, refer to polymeric forms of nucleotides of anylength, either ribonucleotides or deoxyribonucleotides. These termsinclude a single-, double- or triple-stranded DNA, genomic DNA, cDNA,RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidinebases, or other natural, chemically or biochemically modified,non-natural or derivatized nucleotide bases. The backbone of thepolynucleotide can comprise sugars and phosphate groups (as maytypically be found in RNA or DNA), or modified or substituted sugar orphosphate groups. In addition, a double-stranded polynucleotide can beobtained from the single stranded polynucleotide product of chemicalsynthesis either by synthesizing the complementary strand and annealingthe strands under appropriate conditions, or by synthesizing thecomplementary strand de novo using a DNA polymerase with an appropriateprimer. A nucleic acid molecule can take many different forms, e.g., agene or gene fragment, one or more axons, one or more introns, mRNA,cDNA, recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs,uracyl, other sugars and linking groups such as fluororibose andthioate, and nucleotide branches. As used herein, “DNA” or “nucleotidesequence” includes not only bases A, T, C, and G, but also includes anyof their analogs or modified forms of these bases, such as methylatednucleotides, internucleotide modifications such as uncharged linkagesand thioates, use of sugar analogs, and modified and/or alternativebackbone structures, such as polyamides. In a particular embodiment, anucleic acid molecule comprises a nucleotide sequence encoding a variantof SpA, as described herein.

The term “Fc-binding,” “binds to an Fc portion” or “binding to an Fcportion” refers to the ability of an affinity ligand described herein,to bind to the constant part (Fc) of an antibody. In some embodiments, aligand according to the present invention binds an Fc portion of anantibody (e.g., human IgG1, IgG2 or IgG4) with an affinity of at least10⁻⁷ M, or at least 10⁻⁸ M, or at least 10⁻⁹ M.

As used herein, the term “Fab binding” or “binding to a Fab portion”refers to the ability of an affinity ligand described herein, to bind toa Fab region of an antibody or an immunoglobulin molecule. The term“reduced binding to a Fab portion” refers to any decrease in binding toa Fab (or F(ab)₂) portion of an immunoglobulin molecule by a SpA-basedligand according to the present invention relative to a control (e.g., awt SpA domain), where the ligand further includes a mutation in one ormore amino acids. In some embodiments, a ligand according to the presentinvention and its wt counterpart (used as a control) includes theglycine residue at position 29 replaced with an amino acid other thanalanine or tryptophan. In a particular embodiment, a ligand according tothe present invention includes a lysine residue at position 29. In aparticular embodiment, binding to a Fab portion of an immunoglobulinmolecule by a ligand described herein is undetectable using conventionaltechniques in the art and those described herein. Binding to animmunoglobulin molecule can be detected using well known techniquesincluding those described herein and including but not limited to, forexample, affinity chromatography and surface plasmon resonance analysis.In some embodiments, an immunoglobulin binding protein encompassed bythe present invention binds an immunoglobulin molecule with an affinityof at least 10⁻¹⁰M.

The term “N-terminus,” as used herein, refers to amino-terminus of theamino acid sequence of a SpA domain, starting at position 1 or atposition 2 of the amino acid sequence of each of the domains, asdepicted in FIG. 1. However, it is understood that the first amino acidin a sequence may be preceded by a methionine amino acid residue oranother amino acid to facilitate homogenous post translationalprocessing of the protein such as, for example, an alanine, a glycine ora valine. The SpA ligands described herein include a deletion of atleast 3, or at least 4, or at least 5 consecutive amino acids from theN-terminus (starting at position 1 or at position 2 of B, C or Z domainamino acid sequences shown in FIG. 1) of a SpA domain. In other words,such ligands include a deletion of consecutive amino acids 1 through 3,or consecutive amino acids 1 through 4, or consecutive amino acids 1through 5 etc., of SpA domains B, Z or C or such ligands include adeletion of consecutive amino acids 2 through 4, or consecutive aminoacids 2 through 5, or consecutive amino acids 2 through 6 etc., of SpAdomains B, Z or C (amino acid sequences of wt B, C and Z domains aredepicted in FIG. 1, which are modified to include deletions from theN-terminus). In a particular embodiment, a ligand according to thepresent invention includes 5 C domains, with each domain including adeletion of 4 consecutive amino acids from the N-terminus, starting atposition 1.

The amino acid sequence of the B domain of SpA containing a deletion of3 consecutive amino acids from the N-terminus is depleted in SEQ IDNO:13, and that containing a deletion of 4 consecutive amino acids fromthe N-terminus is depicted in SEQ ID NO: 28. Additionally, the aminoacid sequence of the C domain of SpA containing a deletion of 3consecutive amino acids from the N-terminus is depicted in SEQ ID NO:14; and that containing a deletion of 4 consecutive amino acids from theN-terminus is depicted in SEQ ID NO: 29. Further the amino acid sequenceof the Z domain containing a deletion of 3 consecutive amino acids fromthe N-terminus is depicted in SEQ ID NO:15; and that containing adeletion of 4 consecutive amino acids from the N-terminus is depicted inSEQ ID NO: 30.

In general, in case of multimeric forms of SpA-based ligands describedherein, the amino acid sequences of the monomeric forms of the ligandsare simply repeated, as desirable. However, it is to be noted that, incase of some multimeric forms of ligands according to the presentinvention, not all domains need to have a deletion from the N-terminus.For example, in some embodiments, ligands do not contain a deletion inthe N-terminus of the first domain in the multimeric form of ligand;however, subsequent domains in the ligand contain a deletion of at least3 consecutive amino acids from the N-terminus or at least 4 consecutiveamino acids from the N-terminus or at least 5 consecutive amino acidsfrom the N-terminus.

The SpA-based ligands according to the present invention harbor superiorand unexpected properties, i.e., reduced fragmentation during use inpurification processes, as evidenced by the Examples herein. Notably,the teachings in the prior art appear to teach away from the motivationto make and use such ligands. For example, U.S. Patent Publication No.20100048876, discusses a ligand which includes a deletion in amino acidresidues 3 through 6 of the C domain of SpA; however, based on theteachings of this publication (see, e.g., FIG. 2 of U.S. PatentPublication No. 20100048876), it appears that the deletion mutantdescribed therein performs poorly with respect to retention of bindingcapacity relative to the wt C domain of SpA, over extended causticexposure. Accordingly, based on the teachings of this reference, itwould be less desirable to use a deletion mutant of a SpA C domain, whenit loses more binding capacity over time, relative to its wild-typecounterpart.

For the sake of convenience, the various sequences referenced throughthe application are summarized in Table 1 below.

TABLE I Brief Description of Sequence SEQ ID AA—Amino Acid; NA—NucleicAcid; Δ—having a deletion NO: wt E domain AA 1 wt A domain AA 2 wt Bdomain AA 3 wt C domain AA 4 wt D domain AA 5 Z domain AA 6 wt E domainNA 7 wt A domain NA 8 wt B domain NA 9 wt C domain NA 10 wt D domain NA11 Z domain NA 12 B domain Δ 3 AA monomer 13 C domain Δ 3 AA monomer 14Z domain Δ 3 AA monomer 15 B domain Δ 3 AA dimer-both domains havingdeletion 16 C domain Δ 3 AA dimer-both domains having deletion 17 Zdomain Δ 3 AA dimer-both domains having deletion 18 B domain Δ 3 AAdimer-only second domain has deletion 19 C domain Δ 3 AA dimer-onlysecond domain has deletion 20 Z domain Δ 3 AA dimer-only second domainhas deletion 21 B domain Δ 3 AA pentamer-all domains have deletion 22 Cdomain Δ 3 AA pentamer-all domains have deletion 23 Z domain Δ 3 AApentamer-all domains have deletion 24 B domain Δ 3 AA pentamer-firstdomain does not have deletion 25 C domain Δ 3 AA pentamer-first domaindoes not have deletion 26 Z domain Δ 3 AA pentamer-first domain does nothave deletion 27 B domain Δ 4 AA monomer 28 C domain Δ 4 AA monomer 29 Zdomain Δ 4 AA monomer 30 B domain Δ 4 AA dimer-both domains havingdeletion 31 C domain Δ 4 AA dimer-both domains having deletion 32 Zdomain Δ 4 AA dimer-both domains having deletion 33 B domain Δ 4 AAdimer-only second domain has deletion 34 C domain Δ 4 AA dimer-onlysecond domain has deletion 35 Z domain Δ 4 AA dimer-only second domainhas deletion 36 B domain Δ 4 AA pentamer-all domains have deletion 37 Cdomain Δ 4 AA pentamer-all domains have deletion 38 Z domain Δ 4 AApentamer-all domains have deletion 39 B domain Δ 4 AA pentamer-firstdomain does not have deletion 40 C domain Δ 4 AA pentamer-first domaindoes not have deletion 41 Z domain Δ 4 AA pentamer-first domain does nothave deletion 42 wt E domain AA non-Fab (G29K) 43 wt A domain AA non-Fab(G29K) 44 wt B domain AA non-Fab (G29K) 45 wt C domain AA non-Fab (G29K)46 wt D domain AA non-Fab (G29K) 47 Z domain AA non-fab (A29K) 48 wt Edomain NA non-Fab (G29K) 49 wt A domain NA non-Fab (G29K) 50 wt B domainNA non-Fab (G29K) 51 wt C domain NA non-Fab (G29K) 52 wt D domain NAnon-Fab (G29K) 53 Z domain NA non-Fab (A29) 54 B domain Δ 3 AA monomernon-Fab (G29K) 55 C domain Δ 3 AA monomer non-Fab (G29K) 56 Z domain Δ 3AA monomer non-Fab (A29K) 57 B domain Δ 3 AA dimer-both domains havingdeletion non-Fab 58 (G29K) C domain Δ 3 AA dimer-both domains havingdeletion non-Fab 59 (G29K) Z domain Δ 3 AA dimer-both domains havingdeletion non-Fab 60 (A29K) B domain Δ 3 AA dimer-only second domain hasdeletion non-Fab 61 (G29K) C domain Δ 3 AA dimer-only second domain hasdeletion non-Fab 62 (G29K) Z domain Δ 3 AA dimer-only second domain hasdeletion non-Fab 63 (A29K) B domain Δ 3 AA pentamer-all domains havedeletion non-Fab 64 (G29K) C domain Δ 3 AA pentamer-all domains havedeletion non-Fab 65 (G29K) Z domain Δ 3 AA pentamer-all domains havedeletion non-Fab 66 (A29K) B domain Δ 3 AA pentamer-first domain doesnot have deletion non- 67 Fab (G29K) C domain Δ 3 AA pentamer-firstdomain does not have deletion non- 68 Fab (G29K) Z domain Δ 3 AApentamer-first domain does not have deletion non- 69 Fab (A29K) B domainΔ 4 AA monomer non-Fab (G29K) 70 C domain Δ 4 AA monomer non-Fab (G29K)71 Z domain Δ 4 AA monomer non-Fab (A29K) 72 B domain Δ 4 AA dimer-bothdomains having deletion non-Fab 73 (G29K) C domain Δ 4 AA dimer-bothdomains having deletion non-Fab 74 (G29K) Z domain Δ 4 AA dimer-bothdomains having deletion non-Fab 75 (A29K) B domain Δ 4 AA dimer-onlysecond domain has deletion non-Fab 76 (G29K) C domain Δ 4 AA dimer-onlysecond domain has deletion non-Fab 77 (G29K) Z domain Δ 4 AA dimer-onlysecond domain has deletion non-Fab 78 (A29K) B domain Δ 4 AApentamer-all domains have deletion non-Fab 79 (G29K) C domain Δ 4 AApentamer-all domains have deletion non-Fab 80 (G29K) Z domain Δ 4 AApentamer-all domains have deletion non-Fab 81 (A29K) B domain Δ 4 AApentamer-first domain does not have deletion non- 82 Fab (G29K) C domainΔ 4 AA pentamer-first domain does not have deletion non- 83 Fab (G29K) Zdomain Δ 4 AA pentamer-first domain does not have deletion non- 84 Fab(A29K) Z domain dimer non-Fab (A29K) 85 His tag NA 86 Z Domain Δ 1 AAdimer-first domain does not have deletion non-Fab 87 (A29K) Z Domain Δ 2AA dimer-first domain does not have deletion non-Fab 88 (A29K) A DomainΔ 4 AA dimer-first domain has a N-terminal deletion 89 D Domain Δ 4 AAdimer-first domain has a N-terminal deletion 90 Z domain pentamernon-Fab (A29K) 91 C domain dimer AA 92 C domain Δ 4 AA pentamer with thefirst domain having N-terminus alanine non-Fab 93 (G29K) C domain Δ 4 AApentamer with the first domain with N-terminus alanine 94 C domainpentamer non-Fab (G29K) AA 95 C domain pentamer wild type AA 96

II. Generation of SpA-Based Molecules for Use as Chromatography Ligands

The SpA-based affinity chromatography ligands encompassed by the presentinvention can be made using any suitable methods known in the art.

For example, as an initial step, standard genetic engineeringtechniques, e.g., those described in the laboratory manual entitledMolecular Cloning by Sambrook, Fritsch and Maniatis, may be used for thegeneration of nucleic acids which express the SpA ligand moleculesdescribed herein.

In some embodiments, a nucleic acid molecule encoding one or moredomains of SpA having an N-terminus deletion can be cloned into asuitable vector for expression in an appropriate host cell. Suitableexpression vectors are well-known in the art and typically include thenecessary elements for the transcription and translation of the variantSpA coding sequence.

SpA molecules described herein may also be synthesized chemically fromamino acid precursors for fragments using methods well known in the art,including solid phase peptide synthetic methods such as the Boc(tert-butyloxycarbonyl) or Fmoc (9-fluorenylmethyloxy carbonyl)approaches (see, e.g., U.S. Pat. Nos. 6,060,596; 4,879,378; 5,198,531;5,240,680).

Expression of SpA molecules described herein can be accomplished in avariety of cells types such as e.g., eukaryotic host cells such as yeastcells, insect cells and mammalian cells and prokaryotic host cells,e.g., bacteria such as E. coli.

In some embodiments, SpA molecules may be expressed on the surface of abacteriophage such that each phage contains a DNA sequence that codesfor an individual SpA molecule displayed on the phage surface. Theaffinity of the SpA molecule for an immunoglobulin can be readilyassayed for using standard techniques in the art and those describedherein, e.g., ELISA and Biacore™ 2000 standard set up (BIACORE AB,Uppsala Sweden). It is desirable that the binding affinity of a SpAmolecule of the present invention to an immunoglobulin is at leastcomparable with that of the parent molecule, where the molecule exhibitsreduced fragmentation during use, as described herein.

III. Supports Used for the Preparation of Chromatography Matrices

In some embodiments, SpA ligands encompassed by the present inventionare immobilized onto a support, e.g., a solid support or a solublesupport, to generate an affinity chromatography matrix suitable for theseparation of biomolecules such as, e.g., immunoglobulins andFc-containing proteins.

In some embodiments, a ligand according to the present invention isimmobilized onto a solid support. Without wishing to be bound by theory,it is contemplated that any suitable solid support may be used for theattachment of a ligand according to the invention. For example, solidsupport matrices include, but are not limited to, controlled pore glass,silica, zirconium oxide, titanium oxide, agarose, polymethacrylate,polyacrylate, polyacrylamide, polyvinylether, polyvinyl alcohol andpolystyrene and derivatives thereof (e.g., alloys thereof). A solidsupport may be a porous material or a non-porous material.

In some embodiments, a solid support is a porous material. A porousmaterial used as a solid support may be comprised of a hydrophiliccompound, a hydrophobic compound, an oleophobic compound, an oleophiliccompound or any combination thereof. The porous material may becomprised of a polymer or a copolymer. Examples of suitable porousmaterials, include, but are not limited to polyether sulfone, polyamide,e.g., nylon, polysaccharides such as, for example, agarose andcellulose, polyacrylate, polymethacrylate, polyacrylamide,polymethacrylamide, polytetrafluoroethylene, polysulfone, polyester,polyvinylidene fluoride, polypropylene, polyethylene, polyvinyl alcohol,polyvinylether, polycarbonate, polymer of a fluorocarbon, e.g.poly(tetrafluoroethylene-co-perfluoro(alkyl vinyl ether)), glass,silica, zirconia, titania, ceramic, metal and alloys thereof.

The porous material may be comprised of an organic or inorganic moleculeor a combination of organic and inorganic molecules and may be comprisedof one or more functional groups, e.g., a hydroxyl group, an epoxygroup, a thiol group, an amino group, a carbonyl group, or a carboxylicacid group, suitable for reacting, e.g., forming covalent bonds forfurther chemical modification, in order to covalently bind to a protein.In another embodiment, the porous material may not possess a functionalgroup but can be coated with a layer of material that bears functionalgroups such as, a hydroxyl group, a thiol group, an amino acid group, acarbonyl group, or a carboxylic acid group.

In some embodiments, a conventional affinity separation matrix is used,e.g., of organic nature and based on polymers that expose a hydrophilicsurface to the aqueous media used, i.e. expose hydroxy (—OH), carboxy(—COOH), carbonyl (—CHO, or RCO—R′), carboxamido (—CONH₂, possibly inN-substituted forms), amino (—NH₂, possibly in substituted form), oligo-or polyethylenoxy groups on their external and, if present, also oninternal surfaces. In one embodiment, the polymers may, for instance, bebased on polysaccharides, such as dextran, starch, cellulose, pullulan,agarose etc, which advantageously have been cross-linked, for instancewith bisepoxides, epihalohydrins, allyl bromide, allyglycidyl ether,1,2,3-trihalo substituted lower hydrocarbons, to provide a suitableporosity and rigidity. In another embodiment, the solid supportcomprises porous agarose beads. The various supports used in the presentinvention can be readily prepared according to standard methods known inthe art, such as, for example, inverse suspension gelation described,e.g., in Hjerten, Biochim Biophys Acta 79 (2), 393-398 (1964).Alternatively, the base matrices can be commercially available products,such as Sepharose™ FastFlow (GE HEALTHCARE, Uppsala, Sweden). In someembodiments, especially advantageous for large-scale separations, thesupport is adapted to increase its rigidity, and hence renders thematrix more suitable for high flow rates.

Alternatively, the solid support can be based on synthetic polymers,such as polyvinyl alcohol, polyvinylether, polyhydroxyalkyl acrylates,polyhydroxyalkyl methacrylates, polyacrylamides, polymethacrylamidesetc. in case of hydrophobic polymers, such as matrices based on divinyland monovinyl-substituted benzenes, the surface of the matrix is oftenhydrophilized to expose hydrophilic groups as defined above to asurrounding aqueous liquid. Such polymers can be easily producedaccording to standard methods, see e.g., Arshady, Chimicae L'Industria70 (9), 70-75 (1988). Alternatively, a commercially available product,such as Source™ (GE HEALTHCARE, Uppsala, Sweden) and Poros (APPLIEDBIOSYSTEMS, Foster City, Calif.) may be used.

In yet other embodiments, the solid support comprises a support ofinorganic nature, e.g. silica, zirconium oxide, titanium oxide andalloys thereof. The surface of inorganic matrices is often modified toinclude suitable reactive groups for further reaction to SpA and itsvariants. Examples include CM Zirconia (Ciphergen-BioSepra(CERGYPONTOISE, France) and CPG® (MILLIPORE).

In some embodiments, the solid support may, for instance, be based onzirconia, titania or silica in the form of controlled pore glass, whichmay be modified to either contain reactive groups and/or sustain causticsoaking, to be coupled to ligands.

Exemplary solid support formats include, but are not limited to, a bead(spherical or irregular), a hollow fiber, a solid fiber, a pad, a gel, amembrane, a cassette, a column, a chip, a slide, a plate or a monolith.

With respect to the format of a matrix, in one embodiment, it is in theform of a porous monolith, which may be made using an inorganic materialsuch as, e.g., silica, or an organic material such as, e.g.polymethacrylate, polyacrylate, polyacrylamide, polymethacrylamide,polytetrafluoroethylene, polysulfone, polyester, polyvinylidenefluoride, polypropylene, polyethylene, polyvinyl alcohol, polyvinyletherand polycarbonate. In case of a monolith, it may be formed viapolymerization or by coating a substrate.

In an alternative embodiment, the matrix is in beaded or particle formthat can be porous or non-porous. Particles may be spherical ornon-spherical as well as magnetic or non-magnetic. Matrices in beaded orparticle form can be used as a packed bed or in a suspended form.Suspended forms include those known as expanded beds and puresuspensions, in which the particles or beads are free to move. In caseof monoliths, packed bed and expanded beds, the separation procedurecommonly follows conventional chromatography with a concentrationgradient. In case of pure suspension, batch-wise mode will be used.Also, solid support in forms such as a surface, a chip, a capillary, ora filter may be used.

The matrix could also be in the form of membrane in a cartridge. Themembrane could be in flat sheet, spiral, or hollow fiber format.

In another embodiment, a ligand according to the present invention isattached to a soluble support, e.g., a soluble polymer or a watersoluble polymer. Exemplary soluble supports include, but are not limitedto, a bio-polymer such as, e.g., a protein or a nucleic acid. In someembodiments, biotin may be used as a soluble polymer, e.g., as describedin US Patent Publication No. 20080108053. For example, biotin may bebound to a ligand, e.g., a SpA-based ligand according to the presentinvention, which subsequent to being bound to the ligand, can be usedfor isolating a protein of interest, e.g., an antibody or fragmentthereof, e.g., present in a crude mixture and the protein of interestcan be isolated or separated via precipitation of thebiotin-ligand-protein polymer complex in either a reversible orirreversible fashion. The polymer may also be a synthetic solublepolymer, such as, for example, including, but not limited, to a polymercontaining negatively charged groups (carboxylic or sulfonic),positively charged groups (quaternary amine, tertiary amine, secondaryor primary groups), hydrophobic groups (phenyl or butyl groups),hydrophilic groups (hydroxyl, or amino groups) or a combination of theabove. Exemplary synthetic soluble polymers can be found inInternational PCT Publication No. WO2008091740 and U.S. Publication No.US20080255027, the entire teachings of each of which are incorporated byreference herein. These polymers, upon specific physical changes in oneor more conditions such as pH, conductivity or temperature, can be usedto purify the protein of interest via precipitation in either areversible or an irreversible fashion. Synthetic soluble polymers may beused alone or may be coupled with a ligand according to the presentinvention and used for capture/purification of a protein of interestsuch as, e.g., an antibody or a fragment thereof, via precipitation ineither a reversible or an irreversible fashion.

In some embodiments, ligands are attached to a membrane in a multi-wellplate format. In yet other embodiments, the ligands are incorporatedinto a capillary or a microfluidics device.

IV. Methods for Attaching a Ligand to a Support

Any suitable technique may be used for attaching a ligand describedherein to a support, e.g., a solid support including those well-known inthe art and described herein. For example, in some embodiments, theligand may be attached to a support via conventional coupling techniquesutilizing, e.g. amino and/or carboxy groups present in the ligand. Forexample, bisepoxides, epichlorohydrin, CNBr, N-hydroxysuccinimide (NHS)etc., are well-known coupling reagents. In some embodiments, a spacer isintroduced between the support and the ligand, which improves theavailability of the ligand and facilitates the chemical coupling of theligand to the support.

In various embodiments encompassed by the present invention, more thanone site on a ligand is attached to a solid support such (i.e., viamultipoint attachment), thereby resulting in an affinity chromatographymatrix which shows reduced fragmentation of the ligand upon extendedcaustic exposure (both in case of the free ligand as well as theattached ligand).

Attachment of a SpA-based chromatography ligand to a solid support canbe achieved via many different ways known, most of which are well knownin the art, as well as those described herein. See, e.g., Hermanson etal., Immobilized Affinity Ligand Techniques, Academic Press, pp. 51-136(1992).

For example, protein ligands can be coupled to a solid support viaactive groups on either the surface of the solid support or the proteinligand, such as, for example, hydrolxyl, thiol, epoxide, amino,carbonyl, epoxide, or carboxylic acid group. Attachment can be achievedusing known chemistries including, but not limited to, use of cyanogenbromide (CNBr), N-hydroxyl succinimide ester, epoxy (bisoxirane)activation, and reductive amination.

For example, thiol-directed protein coupling has been described in theliterature. See, e.g., Ljungquist, et al. Eur. J. Biochem. Vol 186, pp.558-561 (1989). This technique has been previously applied for couplingSpA to a solid suppose. Since wild type SpA does not contain thiolgroups, the attachment is achieved by recombinantly inserting a thiolcontaining cysteine at the C-terminus of SpA. See, e.g., U.S. Pat. No.6,399,750. Several commercial products such as MabSelect™, MabSelect™Xtra and MabSelect™ SuRe, MabSelect™ SuRe LX are produced via thismechanism. It has been reported that this terminal cysteine only reactswith the epoxide group on the solid surface, thereby resulting in singlepoint attachment of the SpA to the solid support. See, e.g., ProcessScale Bioseparations for the Biopharmaceutical Industry, CRC Press,2006, page 473.

In some embodiments according to the present invention, more than onesite on the SpA-based chromatography ligands is attached to a solidsupport via non-discriminate, multipoint attachment. In general, SpAcontains abundant free amino groups from numerous lysines in eachdomain. The attachment of a SpA domain to a solid support via multipointattachment, e.g., a chromatography resin with epoxide or aldehyde group,can be achieved by reacting the amino group of lysine on SpA, viaepoxide ring-opening or reductive amination, respectively. In certainembodiments, multipoint attachment can be achieved by the reaction ofone or more naturally occurring amino acids on SpA having free hydroxylgroups, such as, for example, serine and tyrosine, with a supportcontaining an epoxide group via a ring-opening reaction. Alternatively,multipoint attachment can be achieved, for example, by the reaction ofnaturally occurring amino acids on SpA having free carboxylic acidgroups, such as, for example, aspartic acid and glutamic acid, with asupport containing amino groups via, for example,N,N′-carbonyldiimidazole. Multipoint attachment of the ligand to supportcan also be achieved by a combination of all the above mechanisms.

SpA-based chromatography ligands may also be attached to a solid supportvia an associative mechanism. For example, an associative group mayinteract with a ligand of interest non-covalently via ionic, hydrophobicor a combination of interactions, thereby to attach ligand of interestonto the solid surface. This facilitates the high efficiency coupling ofligand to the solid matrix, for example, as described in U.S. Pat. Nos.7,833,723 and 7,846,682, incorporated by reference herein, therebyresulting in ligand density higher than that without the associativegroups. Associative groups suitable for use in the invention includecharged species such as ionic species, and uncharged species such ashydrophobic species. The associative group may modify the solid support,e.g. by covalently binding directly with the solid support. Suitableexamples of ionic species may include quaternary amines, tertiaryamines, secondary amines, primary amines, a sulfonic group, carboxylicacid, or any combination thereof. Suitable examples of hydrophobicspecies may include a phenyl group, a butyl group, a propyl group, orany combination thereof. It is also contemplated that mixed mode speciesmay be used. The associative group may also interact with the proteinligand. Thus the interaction between the associative group and theprotein ligand may be comprised of a mixture of interactions, e.g. ionicand hydrophobic species.

The associative group may be covalently coupled to the solid support byreacting a functional group on the solid support with a functional groupon the associative group. Suitable functional groups include, but arenot limited to amines, hydroxyl, sulfhydryl, carboxyl, imine, aldehyde,ketone, alkene, alkyne, azo, nitrile, epoxide, cyanogens and activatedcarboxylic acid groups. As an example, agarose beads contain hydroxylgroups which may be reacted with the epoxide functionality of apositively charged associative group, such as glycidyl trimethylammoniumchloride. A skilled artisan will appreciate that a plurality ofassociative groups may be coupled to the solid support provided that atleast one bifunctional associative group is used. Thus associativegroups may be coupled in tandem to the solid support or they may beindividually coupled directly to the solid support.

In some embodiments, the present invention provides associative groupsand/or protein ligands which may be coupled to a solid support via anintervening linker. The linker may comprise at least one functionalgroup coupled to a linking moiety. The linking moiety may comprise anymolecule capable of being coupled to a functional group. For example,the linking moiety may include any of an alkyl, an alkenyl, or analkynyl group. The linking moiety may comprise a carbon chain rangingfrom 1 to 30 carbon atoms. In some embodiments the linker may becomprised of more than 30 carbon atoms. The linking moiety may compriseat least one hetero-atom such as nitrogen, oxygen and sulfur. Thelinking moiety may be comprised of a branched chain, an unbranched chainor a cyclic chain. The linking moiety may be substituted with two ormore functional groups.

Choosing the appropriate buffer conditions for coupling a protein ligandto a solid support is well within the capability of the skilled artisan.Suitable buffers include, e.g., sodium acetate, sodium phosphate,potassium phosphate, sodium carbonate, sodium bicarbonate, potassiumcarbonate, potassium bicarbonate, sodium chloride, potassium chloride,sodium sulphate, etc. or any combination of the above, with theconcentration ranging from 10 mM to 5M. In some embodiments, theconcentration of salt ranges from 0.1M to 1.5M.

Additional suitable buffers include any non-amine containing buffer suchas carbonate, bicarbonate, sulfate, phosphate and acetate buffers, or acombination of the above. When associative chemistry is used, saltconcentration of the buffer will depend on the associative group used.For example, the salt concentration may be in the range of 5 nM-100 mM.Where a charged species is used, the salt concentration may be at least5 nM but less than 0.1M, at least 5 nM but less than 0.01M, at least 5nM but less than 0.001M. In certain embodiments, the salt concentrationmay be 0.01M. Where a hydrophobic species is used a high saltconcentration is usually desirable. Thus the salt concentration may begreater than 0.001M, greater than 0.01M, or greater than 0.1M.

In some embodiments, when associative chemistry is used, the reaction isperformed at a temperature ranging from 0° C. to 99° C. In certainembodiments the reaction method is practiced at a temperature less than60° C., less than 40° C., less than 20° C., or less than 10° C. In someembodiments the method of the invention is practiced at a temperature ofabout 4° C. In other embodiments the method of the invention ispracticed at a temperature of 20° C.

V. Assaying for Reduced Fragmentation of the Ligands

The present invention provides affinity chromatography matrices whichincorporate SpA ligands based on one or more B, Z or C domains, whereone or more domains include a deletion of 3 or 4 or 5 consecutive aminoacids from the N-terminus, starting at position 1 or at position 2. Insome embodiments, more than one site on an SpA-based ligand is attachedto a chromatography matrix.

The present invention is based on an unexpected and surprising discoverythat the ligands described herein, both in free form as well as whenimmobilized onto a solid support (e.g., a chromatography matrix),exhibit reduced fragmentation following exposure to extended causticconditions during use in purification processes. As discussed above,such fragmentation is undesirable as it leads to potentially immunogenicfragments of SpA domains ending up with the potentially therapeutictarget protein. Further the fragmentation makes the purification processmore costly due to the need to use more ligand during the process.

Fragmentation of affinity ligands can be readily detected using methodsknown in the art and those described herein. Such methods include, butare not limited to, SDS-PAGE and SEC.

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)is commonly used for molecular weight analysis of proteins. SDS is adetergent that dissociates and unfolds proteins. The SDS binds to thepolypeptides to form complexes with fairly constant charge to massratios. The elcetrophoretic migration rate through a gel is thereforedetermined only by the size of the complexes. Molecular weights aredetermined by simultaneously running marker proteins of known molecularweight. The gel is typically stained and the presence of biomolecules ofdifferent molecular weights can be visualized.

Size-exclusion chromatography (SEC) is a method in which molecules insolution are separated by their size. It is usually applied to large ormacromolecular complexes such as proteins and industrial polymers.Detection of different molecular species is typically performed byUV-Vis or by light scattering. In the case of UV-Vis, a wavelengthspecific for detection of certain species is chosen. The orders in whichcertain molecular species elute, as observed on the chromatogram, aswell as the intensity of corresponding peaks, provides information onthe species type as well as relative quantity.

As demonstrated by the examples included herein, the SpA ligandsaccording to the present invention exhibit reduced fragmentationrelative to their wt counterparts, following exposure to causticconditions. In an exemplary experiment to show reduced fragmentation, aSpA ligand having an N-terminus deletion, as described herein, and itswt counterpart, are both exposed to 0.5M NaOH for 25 hrs. The solutionis then neutralized with an acid to pH 7. The neutralized solutions areinjected into SEC or loaded on to an SDS-PAGE gel for analysis andcomparison.

In another exemplary experiment to show reduced fragmentation, anaffinity chromatography matrix including a ligand having an N-terminusdeletion attached to a solid support (immobilized via multipointattachment), as described herein, as well as an affinity chromatographymatrix including its wt counterpart attached to a solid support(immobilized via multipoint attachment), are both exposed to 0.5M NaOHfor 25 hrs. The caustic solution and the matrix (e.g., in the form of aresin) are separated and immediately neutralized with an acid to pH 7.The neutralized solutions are injected into SEC or loaded on to anSDS-PAGE gel for analysis and comparison.

VI. Assaying for Alkaline Stability of the Ligands

In addition to exhibiting reduced fragmentation during use inpurification processes, the ligands described herein are also alkalinestable. Subsequent to the generation of the chromatography matricesincorporating the SpA-based ligands described herein, the alkalinestability of the matrices containing the ligands can be assayed usingstandard techniques in the art and those described herein.

For example, the alkaline stability of a ligand immobilized onto amatrix can be assayed using routine treatment with NaOH at aconcentration of about 0.5M, e.g., as described herein as well as inU.S. Patent Publication No. 20100221844, the entire content of which isincorporated by reference herein in its entirety.

In some embodiments, alkaline stable SpA molecules as well as matricesincorporating the same exhibit an “increased” or “improved” alkalinestability, meaning that the molecules and matrices incorporating thesame are stable under alkaline conditions for an extended period of timerelative to their wt counterparts. Previously, it has been reported thatchromatography matrices incorporating SpA ligands based on the wt B, Cor Z domain of SpA or having a mutation of one or more asparagineresidues provides an improved alkaline stability under conditions wherethe pH is above about 10, such as up to about 13 or 14. However, some ofthese ligands appear to show fragmentation during use, especiallyfollowing repeated cycles of CIP, as observed by the presence offragments on an SDS-PAGE gel or by SEC.

In some embodiments, ligands according to the present invention as wellas matrices incorporating the same are no more alkaline stable thantheir wt counterparts; nonetheless, they exhibit reduced fragmentation.One such ligand described herein is a pentameric form of the C domainligand including an N-terminus deletion in each of the domains andincluding a G29K mutation in each of the domains. Such a ligand mayfurther include an alanine as the very first amino acid in the pentamerto facilitate homogenous post-translational processing.

The present invention is based on the surprising and unexpecteddiscovery of novel SpA ligands (in both free form as well as well whenimmobilized into a chromatography matrix) which exhibit reducedfragmentation during use in purification processes relative to some ofthe previously described ligands, in addition to retaining at least 95%of the initial binding capacity following extended exposure to causticconditions (e.g., 0.1M NaOH for 25 hours or more). In some embodiments,more than one site on the ligands is attached onto a solid support andthese ligands are based on B, C, or Z domains of SpA, where the ligandshave a deletion of 3, 4 or 5 consecutive amino acids from theN-terminus, starting at position 1 or at position 2.

In some embodiments, after 100 cycles, each cycle including a 15 mintreatment with 0.5M NaOH, the percentage of retained binding capacity ofthe SpA ligands described herein (e.g., those comprising one or more B,C or Z domains, and any combinations thereof, where at least one of B, Cor Z domain includes a deletion of at least 3 consecutive amino acidsfrom the N-terminus), is at least 1.25 times more, 1.5 times more, 2.0times more, 2.5 times more, or 3 times more than that of the wtcounterpart.

In one embodiment, the alkaline stability of the immobilized ligand, asassayed by the retention of IgG binding capacity over time, is measuredas follows. The binding capacity, referred to as Qd 50%, is measured byobtaining the volume of IgG loaded to a concentration based onabsorbance at UV_(280mm) of 50% of the initial IgG concentration. Theinitial Qd 50% of the chromatography matrix (e.g., resin packed in acolumn) is measured first. The chromatography matrix (e.g., resin asdescribed above) is then exposed to about 10 cycles of 15 min exposureof 0.5M NaOH at 0.8 mL/min. Qd 50% is measured again. This process isrepeated until the chromatography matrix is exposed to a total of about100 cycles of 0.5M NaOH. Qd 50% is measured one last time and theresults from the affinity chromatography matrix including ligands (e.g.,chromatography resin immobilized with ligands) as described herein arecompared with the respective type wt domains of SpA.

In another assay, caustic or alkaline stability of the matrix ismeasured by static soaking of the matrix. By soaking a measured amountof an affinity chromatography matrix (e.g., in resin format) in 0.1MNaOH, 0.3M NaOH or 0.5M NaOH for 25 hrs with gentle rotation andmeasuring IgG binding capacity before and after the NaOH soaking, thealkaline stability by way of retention of binding capacity of the matrixfor IgG, can be determined.

VII. Methods of Purifying a Target Molecule Using a ChromatographyMatrix of the Invention

In some embodiments, the present invention provides a method ofpurifying a target molecule from a mixture using the affinitychromatography matrices described herein. The target molecule may be anymolecule which is recognized by an affinity ligand provided herein,where the ligand is coupled to a solid support (i.e., a chromatographymatrix). Examples of target molecules include immunoglobulins andFc-containing proteins. The immunoglobulins may be polyclonal antibodiesor a monoclonal antibody or a functional fragment thereof. Functionalfragments include any fragment of an immunoglobulin comprising avariable region that still binds specifically to its antigen while atthe same time retaining its ability to specifically bind to a proteinligand coupled to a solid support.

In some embodiments, a method of isolating a target molecule of interestusing an affinity chromatography matrix described herein includes thesteps of: (a) contacting a solid support including an immobilizedSpA-based chromatography ligand having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 13-42, SEQ ID NOs: 55-84 andSEQ ID NOs: 93-95, with a mixture comprising a target molecule underconditions such that the target molecule specifically binds to theligand; and (b) altering the conditions such that the target molecule isno longer bound to the ligand, thereby isolating the target molecule.

In some embodiments, the altering step includes altering the pH, suchthe target molecule is no longer bound to the ligand. In a particularembodiment, the pH is altered in a manner such that it is more acidicthan the pH conditions in step (a). For example, in one embodiment, step(a) may be performed at a neutral pH, or a pH ranging from about 6 toabout 8 and step (b) may be performed at an acidic pH, e.g., a pHranging from about 1 to about 5.

In another embodiment, step (b) comprises altering the saltconcentration of the buffer in use, such that the target molecule is nolonger bound to the ligand. For example, in one embodiment, a high saltconcentration, e.g., >0.1M, may be used in step (a) and a lower saltconcentration, e.g., <0.1M may be used in step (b). Conversely., in someembodiments, a low salt concentration, e.g., <0.1M may be used in step(a) and a high salt concentration may be used in step (b). In stillother embodiments, both the pH and the salt concentration of the buffermay be altered between step (a) and step (b).

One skilled in the art can readily determine the conditions suitable forbinding a target molecule to a ligand, and thereby alter the conditionsto disrupt the binding of the molecule to the ligand.

In general, it is contemplated that the ligands described herein can beused in any purification process or a purification process train wherenative SpA and recombinant SpA are typically used. In other words, it isgenerally desirable to replace the native SpA (e.g., isolated from S.aureus) and recombinant SpA (e.g., recombinantly expressed wt SpA) inthe current processes in the art with the ligands described herein, inorder to reduce overall cost as well as mitigate the risk of potentiallyimmunogenic SpA fragments co-purifying with a potential therapeuticmolecule.

In some embodiments, the present invention relates to a method ofpurification of antibodies by affinity chromatography, where the methodincludes the following steps: contacting a process feed with an affinitychromatography matrix according to the invention in order to bind one ormore antibodies in the feed; an optional wash step; adding a suitableelution buffer for releasing the bound antibodies from the matrix; andrecovering the one or more antibodies from the eluate. The affinitychromatography matrices described herein may also be used for isolatingantibodies from culture liquids, supernatants as well as fermentationbroths. In case of fermentation broths, the use of affinitychromatography matrices enables the separation of antibodies from hostcell proteins (HCPs), DNA, viruses, endotoxins, nutrients, one or morecomponents of a cell culture medium, e.g., antifoam agents andantibiotics, and product-related impurities, such as misfolded speciesand aggregates.

In a specific embodiment, the feed is subjected to mechanical filtrationbefore it is contacted with the affinity chromatography matrix describedherein, and consequently the mobile phase is a clarified cell culturebroth. Suitable conditions for adsorption are well known to those ofskill in the art.

In another embodiment, the present invention relates to a multi-stepprocess for the purification of antibodies, which process comprises acapture step using an affinity chromatography matrix described hereinfollowed by one or more subsequent steps for intermediate purificationand/or polishing of the antibodies. In a particular embodiment, thecapture step is followed by hydrophobic interaction and/or ion exchangechromatography and/or weak partition chromatography in bind-and elute orflow through mode. In an alternative step, the capture step is followedby multimodal anion or cation exchange chromatography and/or weakpartition chromatography in bind-and-elute or flow through mode.

In another embodiment, any leached SpA-based ligand from the affinitychromatography matrix can be removed by the subsequent purificationsteps to acceptable levels e.g., to levels deemed acceptable for nativeProtein A ligand.

In general, it is contemplated that the ligands described herein may beused in any process which typically employs Protein A ligands.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures, are incorporated herein byreference.

EXAMPLES Example 1 Generation of SpA Ligands With One or More DomainsHaving an N-terminus Deletion of 4 Consecutive Amino Acids

Synthetic genes encoding the following proteins were obtained from DNA2.0 (Menlo Park, Calif.). A SpA dimeric protein containing two Zdomains, each domain containing the mutation at position 29 (A29K) toreduce or eliminate Fab binding (amino acid sequence shown in SEQ ID NO:85); and a SpA dimeric protein containing two Z domains, each domaincontaining the A29K mutation and the second Z domain containing adeletion to delete 4 consecutive amino acids from the N-terminus (aminoacid sequence shown in SEQ ID NO:78).

The 5′ end of each synthetic gene includes a codon for an initiatingmethionine and the 3′ end includes six histidine codons (SEQ ID NO:86)for subsequent purification using NiNTA column. The 5′ and 3′ ends ofeach gene contain NdeI and BamHI restriction sites, respectively. Thesesynthetic genes as well as the expression vector that is used, i.e.,pET11a (EMD), are digested with NdeI and BamHI (NEW ENGLAND BIOLABS,Ipswich, Mass.), the DNA fragments are separated on a 0.7% agarose TAEgel and the appropriate DNA fragments are excised and purified using thegel extraction kit from QIAGEN (Valencia, Calif.). The purified insertsare ligated into the backbone of a pET11a or any other suitableexpression vector using T4 DNA ligase (NEW ENGLAND BIOLABS, Ipswich,Mass.).

The ligation reaction is transformed into DH5□ competent E. coli(INVITROGEN, Carlsbad, Calif.), as per manufacturer's instructions, andplated on Technova LB plates containing 100 g/mL ampicillin and grownovernight at 37° C. In order to obtain purified DNA, individual coloniesare picked for overnight culture in LB containing 100 □g/mL ampicillin.DNA is purified using spin mini-prep kits born QIAGEN (Valencia,Calif.). The identity of recombinant plasmids is confirmed byrestriction digest analysis using NdeI and BamHI (NEW ENGLAND BIOLABS,Ipswich, Mass.). Plasmid maps for the plasmids including both insertedgenes for the Z domain dimeric constructs are shown in FIG. 2.

Additionally, constructs expressing a pentameric form of SpA ligandcontaining 5 Z domains, each domain containing the A29K mutation (aminoacid sequence set forth in SEQ ID NO:91); as well as a pentameric formof SpA ligand containing 5 Z domains, with each domain containing theA29K mutation as well as all but the first domains containing a deletionof 4 consecutive amino acids from the N-terminus (amino acid sequenceset forth in SEQ ID NO:84), are generated.

Further, dimeric SpA ligand constructs containing C domains are alsogenerated. A dimeric construct expressing a C domain ligand isgenerated, the amino acid sequence of which is set forth in SEQ IDNO:92; as well as a dimeric construct expressing a 2 C domain ligand isgenerated, where only the second C domain includes a deletion of 4consecutive amino acids from the N-terminus, the amino acid sequence ofwhich is set forth in SEQ ID NO:35. These C domain dimeric ligands donot have a mutation at position 29.

Example 2 Expression and Purification of SpA-Based Ligands

As discussed above, any suitable bacterial expression system can be usedfor expressing the various SpA ligands described herein. For example,the protein may be expressed in an Escherchia coli strain such as strainBL21(DE3) (PROMEGA, Madison Wis.) using a pET vector such as pET11a(EMD).

A single colony is selected from a plate and grown overnight at 37° C.in LB media containing 100 □g/mL ampicillin. The overnight culture isdiluted 100-fold into fresh LB media containing 100 □g/mL ampicillin andgrown to a cell density such that the optical density at 600 nm is ˜0.8.Following the addition of 1 mM isopropyl-beta-D-thiogalactopyranoside,cells are grown for an additional two hours. Expression is confirmed bySDS-PAGE analysis and Western blotting.

Cells are harvested by centrifugation (4000 rpm, 4° C., 5 minutes) andresuspended in 3 mL of phosphate buffered saline containing 20 mMimidazole. Cells are lysed by sonication, and cell debris is pelleted bycentrifugation (4000 rpm, 4° C., 30 minutes). SpA ligands are purifiedusing NiNTA resin (QIAGEN), applying 25-30 mL cell lysate per 3-mLcolumn. Columns are washed with 30 mL phosphate buffered salinecontaining 20 mM imidazole twice, and SpA is eluted in 3 mL fractions ofphosphate buffered saline containing 200 mM imidazole. SpA is dialyzedovernight into 18 mega-Ohm Milli-Q® water (MILLIPORE, Billerica, Mass.)followed by 10 mM NaHCO₃. Protein concentration is confirmed using theUV spectrometer based on theoretical extinction coefficient (Pace et.al., Protein Science 4:2411 (1995)).

Example 3 Attachment of SpA-Based Ligands to a Solid Support

Subsequent to the generation and expression of various ligands, asdescribed in Examples 1 and 2, they were immobilized via multipointattachment to a solid support.

In an exemplary experiment, agarose resin (Sepharose 4B) (GE HEALTHCARE)is crosslinked using epichlorohydrin according to a previously describedmethod (Porath and Fomstedt, J. Chromatography, 51:479 (1979)). Theagarose resin is subsequently reacted with positively chargedassociative groups, e.g., cations, according to the following method: to10 mL of resin, 5 mL of 75% wt glycidyl trimethylammonium chloride(GTMAC), 5 mL Milli-Q® water (MILLIPORE, Billerica, Mass.) and 0.258 g50% wt sodium hydroxide is added. The reaction vial is rotated in aTechne HB-1D hybridizer (BIBBY SCIENTIFIC, Burlington, N.J.) overnightat room temperature. The resin is then filtered and washed with three10-mL volumes of Milli-Q® water (MILLIPORE, Billerica, Mass.).

The resin (10 mL, filtered cake) is added to a jar containing 3 mL of4.6M NaOH. The mixture is slurried and then 4 mL of butanedioldiglycidyleter (BUDGE) is added. This mixture is rotated at 35° C. forabout 2 hours. The resin is then washed with 5×10 mL of Milli-Q® water(MILLIPORE, Billerica, Mass.) and equilibrated with 2×10 mL of 10 mMNaHCO₃.

Immediately following the BUDGE activation step above, to 5 mL of thefiltered bead cake, 10 mL solution of 10 mM NaHCO₃ containing a 2.5 and2.3 mg/mL concentration of dimeric Z domain ligand containing A29Kmutation (SEQ ID:85) or the dimeric Z domain ligand containingN-terminus deletion in the second Z domain (SEQ ID:78), is added. Themixture is capped in a glass vial and the vial is rotated at 37° C. for2 hours. After two hours, the resin is washed with 3 times with 10 mL ofMilli-Q® water. The filtered bead cake (10 mL) is added to a jarcontaining a 10 mL solution comprised of 1 mL of thioglycerol and 9 mLof a buffer solution with 0.2M NaHCO₃ and 0.5M NaCl. The mixture isslurried and rotated overnight at room temperature. The resin is thenwashed with 3 times with 10 mL of the following buffers: 0.1M Trisbuffer with 0.15M NaCl (pH 8) and 50 mM acetic acid (pH 4.5). This isfollowed by rinsing the resin with 10 mL of Milli-Q® water and 10 mL of20% ethanol water solution (v/v). The final resin is stored in 20%alcohol water solution (v/v) before further use. The method of couplingthe dimeric C domain ligands to a solid support is similar to what isdescribed herein for the dimeric Z domain ligands.

Method of coupling of 5 domain ligands described above (SEQ ID NOs: 91and 84) to agarose base resin is similar to the process above, exceptthat 15 mg/mL of ligand is used during the coupling step. The Z domainpentameric ligands do not contain a His-6 tag.

Example 4 SDS-PAGE Analysis of Supernatants Collected After Caustic Soakof Free or Immobilized Ligands

SDS-PAGE analysis can be used for detecting fragmentation of free andimmobilized ligands described herein, following extended causticexposure. An SDS-PAGE protocol is described below.

SpA in Milli-Q® water, neutralized caustic soaked ligand solution, andneutralized resin soak solutions (each contains ˜0.5 mg/mL of protein)are diluted at 1:1 ratio with Laemunli buffer (BIORAD, Hercules,Calif.). Samples are incubated at 70° C. for 5 minutes to ensureproteins were fully denatured. 10 □L of each sample is loaded to AnyKDgel (BIORAD, Hercules, Calif.) or 15% Tris-HCl Ready gel (BIORAD,Hercules, Calif.). Gel electrophoresis is conducted in 1×Tris-Glycine-SDS running buffer (THERMOFISHER, Waltham, Mass.) at 200volts for 30 minutes. SDS-Gel is then stained in Gelcode Blue stainreagent (THERMOFISHER, Waltham, Mass.) for 1 hour and destained inMilli-Q® water overnight.

Example 5 SEC Analysis of Supernatants Collected After Caustic Soak ofFree or Immobilized Ligands

In addition to SDS-PAGE analysis described above, SEC (size exclusionchromatography) can also be used for fragmentation analysis of the freeand immobilized ligands following extended caustic soak. An SECexperiment is described below.

SEC is conducted on an Agilent 1100 HPLC system (AGILENT, Santa Clara,Calif.). SpA control in Milli-Q® water, neutralized caustic soakedligand solution (˜0.5 mg/mL), and neutralized resin soak solutions arecentrifuged at 13500 RPM for 10 minutes prior to SEC-HPLC analysis.Samples are injected in 20 L onto the SEC column (SEPAX Zenix 7.8 mm×300mm, SEPAX TECHNOLOGIES, INC, Newark, Del.). Sodium phosphate buffer (200mM, pH 7.0) is used as mobile phase with flow rate of 1 mL/min.ChemStation software from Agilent is used for SEC data acquisition andanalysis at both 210 nm and 200 nm.

Example 6 Caustic Soak of Ligands

Following the expression of the ligands, as described in Example 2, theligands are exposed to alkaline conditions.

To 1 mL of each of the ligands described above (at a concentration of 1mg/mL), 1 mL of 1M NaOH is added to a final concentration of 0.5M NaOHand 0.5 mg/mL ligand. The sample is gently rotated for 25 hrs. Thissolution is then neutralized to pH ˜7 using 32 L of glacial acetic acid.

The fragmentation analysis of the Z domain and C domain dimeric ligandsusing SDS-PAGE following with and without caustic soak, is shown in FIG.3. As observed in FIG. 3, the dimeric Z domain ligand (A29K with nodeletions, shown in SEQ ID NO:85, which is used as the control) showssignificant fragmentation following caustic soak in 0.5M NaOH for 25hours, as demonstrated by the presence of a smear as well as presence ofsmaller fragments at around 7 KDa (see Lane 3). In contrast, the dimericZ domain ligand having the A29K mutation as web as a deletion of 4consecutive amino acids in the second domain (amino acid sequence of SEQID NO:78) appears to be largely intact following caustic soak in 0.5MNaOH for 25 hours (see Lane 6).

Similarly, the dimeric C domain ligand having no deletions (the aminoacid sequence set forth in SEQ ID NO:92, used as a control) shows thepresence of a smear as well as smaller fragments around 7 KDa on anSDS-PAGE following caustic soak in 0.5M NaOH for 25 hours (see Lane 9)relative to the dimeric C domain construct which includes a deletion of4 consecutive amino acid deletion from the N-terminus of the seconddomain, the amino acid sequence of which is set forth in SEQ ID NO:35(see Lane 12).

Each of the dimeric Z and C domain ligands additionally includes a His-6tag. The ligands that are not exposed to caustic soak are shown in Lane2 (dimeric Z domain control), Lane 5 (dimeric Z domain ligand having aN-terminus deletion), Lane 8 (dimeric C domain control) and Lane 9(dimeric C domain ligand having a N-terminus deletion).

Reduced fragmentation of the dimeric Z and C domain ligands having anN-terminus deletion, following extended caustic soak, is furtherevidenced by SEC. The results of a representative SEC experiment areshown in FIG. 4 in the form of an SEC chromatogram. As seen in FIG. 4,the controls for the Z domain ligand (having the amino acid sequence setforth in SEQ ID NO:85) as well as the C domain ligand (having the aminoacid sequence set forth in SEQ ID NO:92) show significant fragmentation,identified by arrows on the chromatogram in FIG. 4. In contrast, thedimeric Z and C domain ligands having the N-terminus deletions in thesecond domain (Z domain ligand amino acid sequence is set forth in SEQID NO:78 and the C domain ligand amino acid sequence is set forth in SEQID NO:35), show reduced fragmentation, as identified by boxes on thechromatogram in FIG. 4.

Additionally, the pentameric forms of Z domain ligands described above(i.e., pentameric Z domain ligand having the amino acid sequence of SEQID NO:91 which represents the control, and pentameric Z domain ligandhaving the amino acid sequence of SEQ ID NO:84, which represents thepentameric ligand having a 4 consecutive amino acid N-terminus deletionin all but the first domain) are also analyzed for fragmentation bySDS-PAGE, following caustic soak in 0.5M NaOH for 25 hours.

As evidenced by the SDS-PAGE gel data seen in FIG. 5, the pentamericform of the Z domain ligand having a 4 consecutive amino acid deletionfrom the N-terminus in all but the first domain (the amino acid sequenceof which is set forth in SEQ ID NO:84), shows far less fragmentationfollowing ligand soak in 0.5M NaOH for 25 hours (see Lane 3), relativeto the pentameric Z domain ligand control, the amino acid sequence ofwhich is set forth in SEQ ID NO:91 (see Lane 6). As discussed above,both forms of pentameric ligands have the A29K mutation. The ligandsthat are not soaked appear to be intact (Lanes 2, and 5).

Lanes 8-10 depict the fragmentation observed with a recombinant SpAligand (rSPA), which is routinely used in purification processes. TherSPA ligand appears to show an even far greater degree of fragmentationfollowing caustic soak in 0.5M NaOH for 25 hours relative to the Zdomain control, as observed by a near disappearance of the protein onthe SDS-PAGE (see Lane 9). The rSPA ligand not exposed to causticconditions is in Lane 8.

This result appears to suggest that the Z domain based ligands havingthe N-terminus deletion, as described herein, are far superiorcandidates than the routinely used SpA ligands such as, e.g., rSPA.

A reduction in fragmentation following caustic soak in 0.5M NaOH for 25hours observed with the pentameric Z domain ligand having the N-terminaldeletion is further confirmed using SEC, the results of one suchrepresentative experiment are shown in FIG. 6.

As demonstrated by the chromatogram in FIG. 6, the pentameric form ofthe Z domain control (SEQ ID NO:91) with no amino acid deletion showswell resolved peaks at lower molecular weight, indicating the presenceof smaller fragments. In contrast, the pentameric form of the Z domainhaving the N-terminus deletion (SEQ ID NO:84) shows significantly fewerdistinct peaks at lower intensity, indicating a far less degree offragmentation, relative to the control.

The routinely used SPA ligand, rSPA, shows the most fragmentation orbreakdown with no intact molecule left at all. Notably, the SECchromatogram is consistent with the results of the SDS-PAGE analysis inFIG. 5, further evidencing that the rSPA ligand has degraded so muchthat no significant fragments can be observed on an SEC chromatogramfollowing extended exposure to caustic conditions (i.e., soaking in 0.5MNaOH for 25 hours).

Example 7 Caustic Soak of Ligands Immobilized on Resin

The various ligands described in the foregoing examples are evaluatedfor fragmentation following caustic exposure subsequent to theirattachment to a solid support (e.g., an agarose chromatography resin).

For each resin of interest, 1 mL resin in 5 mL disposable chromatographycolumn (EVERGREEN SCIENTIFIC, Los Angeles, Calif.) is measured usingMilli-Q® water. The resin is conditioned in a column with 2 CV (2 mL) of0.5M NaOH quickly, re-slurried and vacuumed. After repeating the NaOHcondition one more time, the vacuumed wet cake of resin is transferredto 4 mL test tubes (THERMOFISHER, Waltham, Mass.). 2 mL of 0.5M NaOH isadded to the column (bottom is capped) and immediately transferred intothe test tube with the corresponding resin. Place capped test tubes ontoa rotator and rotate the test tubes for 25 hrs. At the end of thecaustic soak, content in the test tubes is poured into a disposablecolumn and the filtrate is collected. Filtrate in 1.5 mL is neutralizedwith 50 μL of glacial acidic acid and is ready for further analysis bySEC and SDS-PAGE.

The SDS-PAGE analysis of the immobilized dimeric Z and C domain ligandsfollowing extended caustic soak (e.g., 0.5M NaOH soak for 25 hours) isshown in FIG. 3. In general, it is expected that if a ligand is causticstable following its immobilization onto a chromatography matrix (e.g.,an agarose resin), that it will not show any significant fragmentation.

As observed by the SDS-PAGE gel of FIG. 3, both the dimeric Z and Cdomain ligands immobilized controls (amino acid sequences set forth inSEQ ID NO:85 and 92, respectively and represented by Lanes 4 and 10 ofthe SDS-PAGE gel, respectively) as well as the dimeric Z and C domainimmobilized ligands containing an N-terminus deletion in the seconddomain (amino acid sequences set forth in SEQ ID NO:78 and 35,respectively and represented by Lanes 7 and 13, respectively), do notappear to show any detectable fragmentation following 0.5M NaOH soak for25 hours, implying that they are both caustic stable.

The SDS-PAGE analysis of the immobilized pentameric Z domain ligandsfollowing extended caustic soak (e.g., 0.5M NaOH soak for 25 hours) isshown in FIG. 5. As observed by the SDS-PAGE gel in FIG. 5, thepentameric Z domain ligand containing an N-terminus deletion in all butthe first domain (amino acid sequence set forth in SEQ ID NO:84, andrepresented by Lane 4 of the SDS-PAGE gel), shows far less fragmentationas compared to its type wt pentameric Z domain control (SEQ ID NO:91 andLane 7). This result suggests that the immobilized pentameric Z domainligand having the N-terminus deletions is more caustic stable comparedto the immobilized pentameric Z domain which does not nave suchdeletions.

Further, the fragmentation of a routinely used ligand (i.e., rSPA) isalso investigated by SDS-PAGE following its immobilization on an agarosechromatography resin and subjecting the resin with the ligand to anextended soak in 0.5M NaOH for 25 hours. As seen in Lane 10 of theSDS-PAGE gel of FIG. 5, the immobilized rSPA shows significantfragmentation following 0.5M NaOH soak for 25 hours, implying that it isnot very caustic stable, relative to the pentameric Z domain ligands(Lanes 4 and 7).

Based on the SDS-PAGE gel results on FIG. 5, it can be concluded thatthe immobilized pentameric Z domain ligand with the N-terminus deletionshas the least fragmentation following extended caustic exposure, andtherefore, is most caustic stable, as compared to the immobilizedpentameric Z domain control ligand and the immobilized rSPA.

Further confirmation of the SDS-PAGE results with the immobilizedpentameric Z domain ligands and the rSPA ligand is obtained by SECanalysis. The results of a representative experiment are depicted in thechromatogram shown in FIG. 7. As seen in FIG. 7, the immobilized Zdomain ligand of SEQ ID NO:84 shows reduced fragmentation followingextended caustic soak, as shown by a box, relative to its wt counterpartof SEQ ID NO:91, which shows resolved lower molecular weight peaks onthe chromatogram. Further, as expected, the immobilized rSPA showsextensive fragmentation following extended caustic soak as observed bybroad and unresolved peaks on the chromatogram.

Example 8 Measurement of Static Binding Capacity of ChromatographyMatrices to an Immunoglobulin Before and After Exposure to 0.5M NaOH for25 Hours

The affinity chromatography matrices (i.e., resins having theimmobilized resins thereon via multipoint attachment) described aboveare further tested for their static binding capacity before and afterexposure to 0.5M NaOH for 25 hours.

In one experiment, each of the chromatography matrices (in 1 mL volume)immobilized with the dimeric or pentameric Z or C domain ligandsdescribed above, either with exposure to 0.1, 0.3 or 0.5M NaOH orwithout exposure to NaOH, is made into 10% slurry in Milli-Q® water(MILLIPORE, Billerica, Mass.). 1 mL of each slurry is added to 15 mL ofpolyclonal IgG (SERACARE, 1 mg/mL)) in 10 mM phosphate saline buffer androtated for 4 hours at room temperature. The reduction of UV at 280 nmis used to calculate capacity before and after caustic binding capacity.The percentage of retained IgG binding capacity is calculated bydividing the IgG binding capacity after caustic exposure by that withoutcaustic exposure. Table II summarizes the results of one suchexperiment. As summarized in Table II, the dimeric Z domain ligand ofSEQ ID NO:78 appears to exhibit a higher retained binding capacityrelative to its wt counterpart (i.e., the dimeric Z domain ligand of SEQID NO:85), following extended caustic soak in 0.5M NaOH for 25 hours.

Similarly, as also summarised in Table II below, the dimeric C domainligand of SEQ ID NO:35 appears to exhibit a higher retained bindingcapacity than its wt counterpart of SEQ ID NO:92, following extendedcaustic soak in 0.5M NaOH for 25 hours.

TABLE II Sequence of Ligand Retained IgG static binding immobilized onmatrix capacity of matrix (%) SEQ ID NO: 85 64 SEQ ID NO: 78 70 SEQ IDNO: 92 65 SEQ ID NO: 35 70

In a further experiment, the IgG binding capacity of a pentameric Zdomain ligand is evaluated following extended caustic soak in 0.1M NaOH,0.3M NaOH or 0.5M NaOH for 25 hours. The results of one such experimentare summarised in Table III below.

Table III shows the percentage of retained IgG binding capacity of amatrix having immobilized thereon a pentameric form of Z domain ligandwhich contains an A29K mutation and all but the first domain include adeletion of four consecutive amino acids from the N-terminus (amino acidsequence shown in SEQ ID NO: 84), after soaking the matrix in 0.1M NaOH,0.3 M NaOH or 0.5M NaOH. As summarized below, the matrix with thepentameric Z domain N-terminus deletion ligand shows up to 95% of theinitial binding capacity after 0.1M NaOH soak for 25 hours; up to 85% ofthe initial binding capacity after 0.3M NaOH soak for 25 hours and up to65% of the initial binding capacity after 0.5M NaOH soak for 25 hours.

Retained IgG static binding capacity of matrix NaOH concentration (M)immobilized with SEQ ID 84 (%) 0.1 95 0.3 85 0.5 65

Example 9 SpA Capture of IgG Before and After Exposure to 0.5M NaOH

In this experiment, purification of a polyclonal immunoglobulin in nullCHO-S feed using a matrix immobilized with a pentameter of Z domainligand with all but the first domain having a 4 consecutive amino aciddeletion is examined along with that having a recombinantly synthesizedSpA (rSPA) in order to demonstrate that the ligands according to thepresent invention work just as well as the recombinant SpA in removingimpurities.

Resin samples immobilized with rSPA (REPLIGEN, Waltham, Mass.), and theZ domain pentameric ligand (amino acid sequence shown in SEQ ID NO:84),are each packed into a chromatography column with 1 cm diameter and 5 cmpacked bed height. After equilibration with phosphate saline buffer (10mM sodium phosphate), the packed resins are subjected to exposure ofnull CHO feed with polyclonal hIgG (SERACARE, 5 mg/mL) at a flow rate of50 cm/hr. After loading at 90% of 5% breakthrough, the resin is washedwith PBS buffer and 50 mM NaOAc, pH 5.5. The bound IgG is subsequentlyeluted with 50 mM NaOAc, pH 3. Fractions are collected and analyzed forimpurity analysis. The packed resin is then exposed to 0.5M NaOH for 15minutes (flow rate 100 cm/hr) before contacting again with polyclonalhIgG in null CHO feed. Resins are then washed with PBS buffer and 50 mMNaOAc, pH 5.5 and IgG is eluted for further assay.

This caustic exposure and feed run cycle is repeated to collect enoughIgG for the subsequent cation exchange step. Leached protein A isquantified using n-Protein A ELISA (REPLIGEN, Waltham, Mass.) accordingto instructions from the manufacturer. Host cell protein is detectedusing the 3G CHO HCP ELISA kit (CYGNUS TECHNOLOGIES, Southport, N.C.),as per the manufacturer's instructions. DNA is detected using Quant-iT™PicoGreen® dsDNA Reagent (LIFE TECHNOLOGIES, Foster City, Calif.). Theresults of one such representative experiment are shown in Table IV.

Example 10 Clearance of Leached SpA Ligands and Further Removal of DNAand Host Cell Protein using Cation Exchange and Anion ExchangeChromatography

The clearance of the leached ligands as well as further removal of hostcell proteins (HCP) and DNA from the elation pool of chromatographyaffinity matrices incorporating either the SpA ligands according to thepresent invention or those containing recombinant SpA, rSPA (REPLIGEN,Waltham, Mass.), is examined as follows.

Combination of elution pools from several repetitions of the experimentdescribed in Example 8 provides the feed for further clearance ofleached ligands and other impurities using cation exchangechromatography.

Fractogel SO₃ ⁻ (MILLIPORE, Billerica, Mass.) is packed into a columnwith bed dimension of 1.0 cm (i.d.)×7 cm (bed height). The column isequilibrated with 50 mM NaOAc pH 4.5, 4 mS/cm and loaded with the pooledIgG from Protein A elution at 140 cm/hr. After column wash with EQbuffer, IgG is eluted with 0.5N NaCl in 50 mM NaOAc over 20 columnvolume (linear gradient). The elution pools are collected in 10 mLfractions and analyzed for leached ligands, DNA, and host cell protein.

The fractions from Fractogel SO₃ ⁻ column are further pooled andadjusted to pH 7.6 at 12 mS/cm. This feed is loaded onto apre-equilibrated (Tris, 25 mM, pH 7.6, ˜1 mS/cm) ChromaSorb device (0.08mL, MILLIPORE, Billerica, Mass.) at flow rate of 1 mL/min. Fractions arecollected for every 187 column volume and further analyzed for leachedligand and host cell protein, as described in Example 8.

As summarized in Table IV below, both the leached rSPA ligand as well asthe pentameric form of Z domain with all but the first domain having aN-terminus deletion of four consecutive amino acids (amino acid sequenceset forth in SEQ ID:84), can be cleared to less than 1 PPM after cationexchange and anion exchange chromatography. In addition, the removal ofhost cell proteins and DNA meets industry standard and is more or lessequivalent in both cases, as also summarized in Table below.

TABLE IV Ligand on resin rSPA SEQ ID: 84 Leached Protein A Protein Apool 7.1 3.8 (PPM) Cation exchange pool 1.1 1.1 Anion exchange pool@ 0.61.0 1 g/mL loading) Host cell proteins Feed 25568 25568 (PPM) Protein Apool 232 133 Cation exchange pool 49 60 Anion exchange pool 3 7 @ 1 g/mLloading) DNA (PPM) Feed 6.8 6.8 Protein A pool 0.05 0.04 Cation exchangepool 0.03 0.03 Anion exchange pool @ Below Below 1 g/mL loading)detection detection limit limit

Example 11 Effect of the Number of N-Terminus Amino Acid Deletions onFragmentation of Ligand Following Extended Caustic Soak

In another experiment, 1, 2, 3 or 4 amino acid residues were deletedfrom the N-terminus of the second domain of a dimeric Z domain ligand,starting at position 1, and the effect of the 1, 2, 3 or 4 amino aciddeletions on the fragmentation of the ligand following extended causticsoak was determined, as compared to the control dimeric Z domain ligand(A29K)

The results of one such experiment are depicted in the chromatogramshown in FIG. 8. As demonstrated in FIG. 8, the effect on fragmentationof the number of amino acid residues that were deleted from theN-terminus of the second domain of the dimeric Z domain ligand can beobserved following extended caustic soak of each of the ligands in 0.5MNaOH for 25 hours followed by SEC analysis, as described in Example 5.

After soaking the ligands in 0.5M NaOH for 25 hours, each of the controlligand (SEQ ID NO:85), the ligand with only the first amino acid deletedfrom the N-terminus of the second domain (SEQ ID NO:87), and the ligandwith the first two amino acids deleted from the N-terminus of the seconddomain (SEQ ID NO:88) shows fragments at lower molecular weight, asdepicted by the arrows, evidencing fragmentation. Whereas, the ligandwith the first three amino acids deleted from the N-terminus of thesecond domain (SEQ ID NO:69) and the ligand with the first four aminoacids deleted from the N-terminus of the second domain (SEQ ID NO:78)showed significantly reduced fragmentation at lower molecular weight asdepicted by boxes in the chromatogram, evidencing a reducedfragmentation.

These results suggest that the affinity ligands based on one or moredomains of Protein A and having at least 3 amino acids deleted from theN-terminus of one or more domains exhibit reduced fragmentationfollowing extended caustic exposure and accordingly, are superiorcandidates for use as affinity chromatography ligands.

Example 12 Retained Binding Capacity Comparison of N-Terminus Deletionand Wild Type C Domain Pentamers, Both Having a Non-Fab Mutation (G29K)

In this experiment, the retained binding capacity of two C domainpentameric ligands immobilized onto a polyvinyl alcohol basedchromatography matrix is examined, one ligand having an N-terminusdeletion (starting at position 1) of 4 amino acids in each of the 5domains, an alanine as the very first amino acid of the pentamericsequence in order to facilitate homogeneous post-translationalprocessing as well as the G29K mutation (the amino acid sequence ofwhich is shown in SEQ ID NO:93) and the other ligand corresponding toits wt counterpart with the G29K mutation (the amino acid sequence ofwhich is shown in SEQ ID NO:95).

The ligands are immobilized onto polyvinyl alcohol based affinitychromatography resins via multipoint attachment (see, e.g., Hermanson etal., Immobilized Affinity Ligand Techniques, Academic Press, pp. 51-136(1992)), and tested for retained dynamic binding capacity upon repeatedNaOH exposure.

In one experiment, the chromatography matrices are packed into columns(0.66 cm i.d.×1.0 cm bed height) and are subjected to a standardchromatographic run with equilibration followed by application of 30 mgpolyclonal human IgG (hIgG) at 60 cm/hr. After extensive washing-out ofunbound proteins with equilibration buffer (10 mM phosphate buffersaline), bound IgG is eluted with elution buffer (0.1M citric acid, pH3) at 60 cm/hr. This is followed by Cleaning-In-Place (CIP) with 0.7MNaOH for 30 mins. The column is re-equilibrated and the run is repeatedfor 16 more times (a cumulative exposure to 0.7M NaOH for 8 hrs).Retained binding capacity is measured by determining total amount ofeluted IgG (elution volume multiplied by IgG concentration measured atUV₂₈₀) over time. Relative retained capacity is plotted against thefirst run with 0 min exposure to NaOH and is shown in FIG. 9. Thisexperiment is repeated 3 times with similar results. As demonstrated inFIG. 9, both C domain pentameric ligands, with and without the deletion,show similar retained binding capacity following extended exposure toNaOH over time. Further, in another experiment, retained bindingcapacity of the C domain pentameric ligand without the alanine andhaving the G29K mutation (amino acid sequence of which is shown in SEQID NO:80) is compared to its wt counterpart with the G29K mutation(amino acid sequence of which is shown in SEQ ID NO:95), with a similarresult (data not shown).

The specification is most thoroughly understood in light of theteachings of the references cited within the specification which arehereby incorporated by reference. The embodiments within thespecification provide an illustration of embodiments in this inventionand should not be construed to limit its scope. The skilled artisanreadily recognizes that many other embodiments are encompassed by thisinvention. All publications and inventions are incorporated by referencein their entirety. To the extent that the material incorporated byreference contradicts or is inconsistent with the present specification,the present specification will supercede any such material. The citationof any references herein is not an admission that such references areprior art to the present invention.

Unless otherwise indicated, all numbers expressing quantities ofingredients, cell culture, treatment conditions, and so forth used inthe specification, including claims, are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessotherwise indicated to the contrary, the numerical parameters areapproximations and may vary depending upon the desired properties soughtto be obtained by the present invention. Unless otherwise indicated, theterm “at least” preceding a series of elements is to be understood torefer to every element in the series. Those skilled in the art willrecognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed by the following claims.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only and are not meant to be limiting in anyway. It is intended that the specification and examples be considered asexemplary only, with a true scope and spirit of the invention beingindicated by the following claims.

What is claimed is:
 1. A method of purifying one or more immunoglobulinsfrom a sample, the method comprising the steps of: a. providing a samplecomprising one or more immunoglobulins; b. contacting the sample with amatrix under conditions such that the one or more immunoglobulins bindto the matrix, wherein the matrix comprises an affinity chromatographyligand attached to a solid support and wherein the ligand is based ontwo or more B domains or two or more Z domains or two or more C domainsof Staphylococcus aureus Protein A, each domain having a deletion of atleast 3 or 4 consecutive amino acids from the N-terminus starting atposition 1 or 2 corresponding to wild-type B, Z or C domain positionsand further having a mutation to reduce Fab binding; and c. recoveringthe one or more bound immunoglobulins by elution.
 2. The method of claim1, wherein the sample is selected from the group consisting of a cellculture liquid, a cell culture supernatant and a fermentation broth. 3.The method of claim 1, wherein the mutation to reduce Fab bindingcomprises the replacement of glycine amino acid residue at position 29with a lysine amino acid residue in case of the B domain and the Cdomain and the replacement of the an alanine amino acid residue atposition 29 with a lysine amino acid residue in case of the Z domain. 4.The method of claim 1, wherein the ligand exhibits reduced fragmentationcompared to its wild-type counterpart following exposure to 0.5M NaOHfor at least 5 hours.
 5. The method of claim 1, wherein the matrixretains at least 95% of its initial binding capacity after 5 hoursincubation in 0.5M NaOH.
 6. The method of claim 1, wherein the matrixretains at least 95% of its initial binding capacity after 25 hoursincubation in 0.1M NaOH.
 7. A method of separating an immunoglobulinfrom one or more of host cell proteins (HCPs), DNA, viruses, endotoxins,nutrients, one or more components of a cell culture medium and productrelated impurities, the method comprising the steps of: a. providing asample comprising an immunoglobulin and one or more of host cellproteins (HCPs), DNA viruses, endotoxins, nutrients, one or morecomponents of a cell culture medium and product related impurities; b.contacting the sample with a matrix under conditions such that theimmunoglobulin binds to the matrix, wherein the matrix comprises anaffinity chromatography ligand attached to a solid support and whereinthe ligand is based on two or more B domains or two or more Z domains ortwo or more C domains of Staphylococcus aureus Protein A, each domainhaving a deletion of at least 3 or 4 consecutive amino acids from theN-terminus starting at position 1 or 2 corresponding to wild-type B, Zor C domain positions and further having a mutation to reduce Fabbinding; and c. recovering the bound immunoglobulin by elution, therebyto separate the immunoglobulin from one or more of host cell proteins(HCPs), DNA, viruses, endotoxins, nutrients, one or more components of acell culture medium and product related impurities.
 8. The method ofclaim 7, wherein the sample is a fermentation broth.
 9. The method ofclaim 7, wherein the mutation to reduce Fab binding comprises thereplacement of glycine amino acid residue at position 29 with a lysineamino acid residue in case of the B domain and the C domain and thereplacement of the an alanine amino acid residue at position 29 with alysine amino acid residue in case of the Z domain.
 10. The method ofclaim 7, wherein one or more components of a cell culture medium areselected from the group consisting of antifoam agents and antibiotics.11. The method of claim 7, wherein the product related impuritiescomprise misfolded species and aggregates.
 12. The method of claim 1,wherein the sample is a clarified cell culture broth.
 13. The method ofclaim 7, wherein the sample is a clarified cell culture broth.
 14. Themethod of claim 7, wherein the matrix retains at least 95% of itsinitial binding capacity after 5 hours incubation in 0.5M NaOH.
 15. Themethod of claim 7, wherein the matrix retains at least 95% of itsinitial binding capacity after 25 hours incubation in 0.1 M NaOH. 16.The method of claim 7, wherein the ligand exhibits reduced fragmentationcompared to its wild-type counterpart following exposure to 0.5M NaOHfor at least 5 hours.